Differences in RAD51 transcriptional response and cell cycle dynamics reveal varying sensitivity to DNA damage among Arabidopsis thaliana root cell types.

Throughout their lifecycle, plants are subjected to DNA damage from various sources, both environmental and endogenous. Investigating the mechanisms of the DNA damage response (DDR) is essential to unravel how plants adapt to the changing environment, which can induce varying amounts of DNA damage. Using a combination of whole-mount single-molecule RNA fluorescence in situ hybridization (WM-smFISH) and plant cell cycle reporter lines, we investigated the transcriptional activation of a key homologous recombination (HR) gene, RAD51, in response to increasing amounts of DNA damage in Arabidopsis thaliana roots. The results uncover consistent variations in RAD51 transcriptional response and cell cycle arrest among distinct cell types and developmental zones. Furthermore, we demonstrate that DNA damage induced by genotoxic stress results in RAD51 transcription throughout the whole cell cycle, dissociating its traditional link with S/G2 phases. This work advances the current comprehension of DNA damage response in plants by demonstrating quantitative differences in DDR activation. In addition, it reveals new associations with the cell cycle and cell types, providing crucial insights for further studies of the broader response mechanisms in plants.

Convergence and molecular evolution of floral fragrance after independent transitions to self-fertilization.

Studying the independent evolution of similar traits provides valuable insights into the ecological and genetic factors driving phenotypic evolution.1 The transition from outcrossing to self-fertilization is common in plant evolution2 and is often associated with a reduction in floral attractive features such as display size, chemical signals, and pollinator rewards.3 These changes are believed to result from the reallocation of the resources used for building attractive flowers, as the need to attract pollinators decreases.2,3 We investigated the similarities in the evolution of flower fragrance following independent transitions to self-fertilization in Capsella.4,5,6,7,8,9 We identified several compounds that exhibited similar changes in different selfer lineages, such that the flower scent composition reflects mating systems rather than evolutionary history within this genus. We further demonstrate that the repeated loss of ß-ocimene emission, one of the compounds most strongly affected by these transitions, was caused by mutations in different genes. In one of the Capsella selfing lineages, the loss of its emission was associated with a mutation altering subcellular localization of the ortholog of TERPENE SYNTHASE 2. This mutation appears to have been fixed early after the transition to selfing through the capture of variants segregating in the ancestral outcrossing population. The large extent of convergence in the independent evolution of flower scent, together with the evolutionary history and molecular consequences of a causal mutation, suggests that the emission of specific volatiles evolved as a response to changes in ecological pressures rather than resource limitation.

Separating phases of allopolyploid evolution with resynthesized and natural Capsella bursa-pastoris.

Allopolyploidization is a frequent evolutionary transition in plants that combines whole-genome duplication (WGD) and interspecific hybridization. The genome of an allopolyploid species results from initial interactions between parental genomes and long-term evolution. Distinguishing the contributions of these two phases is essential to understanding the evolutionary trajectory of allopolyploid species. Here, we compared phenotypic and transcriptomic changes in natural and resynthesized Capsella allotetraploids with their diploid parental species. We focused on phenotypic traits associated with the selfing syndrome and on transcription-level phenomena such as expression-level dominance (ELD), transgressive expression (TRE), and homoeolog expression bias (HEB). We found that selfing syndrome, high pollen, and seed quality in natural allotetraploids likely resulted from long-term evolution. Similarly, TRE and most down-regulated ELD were only found in natural allopolyploids. Natural allotetraploids also had more ELD toward the self-fertilizing parental species than resynthesized allotetraploids, mirroring the establishment of the selfing syndrome. However, short-term changes mattered, and 40% of the cases of ELD in natural allotetraploids were already observed in resynthesized allotetraploids. Resynthesized allotetraploids showed striking variation of HEB among chromosomes and individuals. Homoeologous synapsis was its primary source and may still be a source of genetic variation in natural allotetraploids. In conclusion, both short- and long-term mechanisms contributed to transcriptomic and phenotypic changes in natural allotetraploids. However, the initial gene expression changes were largely reshaped during long-term evolution leading to further morphological changes.

Testing the effect of individual scent compounds on pollinator attraction in nature using quasi-isogenic Capsella lines.

PREMISE: Floral scent, usually consisting of multiple compounds, is a complex trait, and its role in pollinator attraction has received increasing attention. However, disentangling the effect of individual floral scent compounds is difficult due to the complexity of isolating the effect of single compounds by traditional methods. METHODS: Using available quasi-isogenic lines (qILs) that were generated as part of the original mapping of the floral scent volatile-related loci CNL1 (benzaldehyde) and TPS2 (ß-ocimene) in Capsella, we generated four genotypes that should only differ in these two compounds. Plants of the four genotypes were introduced into a common garden outside the natural range of C. rubella or C. grandiflora, with individuals of a self-compatible C. grandiflora line as pollen donors, whose different genetic background facilitates the detection of outcrossing events. Visitors to flowers of all five genotypes were compared, and the seeds set during the common-garden period were collected for high-throughput amplicon-based sequencing to estimate their outcrossing rates. RESULTS: Benzaldehyde and ß-ocimene emissions were detected in the floral scent of corresponding genotypes. While some pollinator groups showed specific visitation preferences depending on scent compounds, the outcrossing rates in seeds did not vary among the four scent-manipulated genotypes. CONCLUSIONS: The scent-manipulated Capsella materials constructed using qILs provide a powerful system to study the ecological effects of individual floral scent compounds under largely natural environments. In Capsella, individual benzaldehyde and ß-ocimene emission may act as attractants for different types of pollinators.

Whole-mount smFISH allows combining RNA and protein quantification at cellular and subcellular resolution.

Multicellular organisms result from complex developmental processes largely orchestrated through the quantitative spatiotemporal regulation of gene expression. Yet, obtaining absolute counts of messenger RNAs at a three-dimensional resolution remains challenging, especially in plants, owing to high levels of tissue autofluorescence that prevent the detection of diffraction-limited fluorescent spots. In situ hybridization methods based on amplification cycles have recently emerged, but they are laborious and often lead to quantification biases. In this article, we present a simple method based on single-molecule RNA fluorescence in situ hybridization to visualize and count the number of mRNA molecules in several intact plant tissues. In addition, with the use of fluorescent protein reporters, our method also enables simultaneous detection of mRNA and protein quantity, as well as subcellular distribution, in single cells. With this method, research in plants can now fully explore the benefits of the quantitative analysis of transcription and protein levels at cellular and subcellular resolution in plant tissues.

Expression pattern of resynthesized allotetraploid Capsella is determined by hybridization, not whole-genome duplication.

Polyploidization, the process leading to the increase in chromosome sets, is a major evolutionary transition in plants. Whole-genome duplication (WGD) within the same species gives rise to autopolyploids, whereas allopolyploids result from a compound process with two distinct components: WGD and interspecific hybridization. To dissect the instant effects of WGD and hybridization on gene expression and phenotype, we created a series of synthetic hybrid and polyploid Capsella plants, including diploid hybrids, autotetraploids of both parental species, and two kinds of resynthesized allotetraploids with different orders of WGD and hybridization. Hybridization played a major role in shaping the relative expression pattern of the neo-allopolyploids, whereas WGD had almost no immediate effect on relative gene expression pattern but, nonetheless, still affected phenotypes. No transposable element-mediated genomic shock scenario was observed in either neo-hybrids or neo-polyploids. Finally, WGD and hybridization interacted and the distorting effects of WGD were less strong in hybrids. Whole-genome duplication may even improve hybrid fertility. In summary, while the initial relative gene expression pattern in neo-allotetraploids was almost entirely determined by hybridization, WGD only had trivial effects on relative expression patterns, both processes interacted and had a strong impact on physical attributes and meiotic behaviors.

The plant-specific DDR factor SOG1 increases chromatin mobility in response to DNA damage.

Homologous recombination (HR) is a conservative DNA repair pathway in which intact homologous sequences are used as a template for repair. How the homology search happens in the crowded space of the cell nucleus is, however, still poorly understood. Here, we measure chromosome and double-strand break (DSB) site mobility in Arabidopsis thaliana, using lacO/LacI lines and two GFP-tagged HR reporters. We observe an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. This increase in mobility is lost in the sog1-1 mutant, a central transcription factor of the DNA damage response in plants. Also, DSB sites show particularly high mobility levels and their enhanced mobility requires the HR factor RAD54. Our data suggest that repair mechanisms promote chromatin mobility upon DNA damage, implying a role of this process in the early steps of the DNA damage response.

Characterization of germline development and identification of genes associated with germline specification in pineapple.

Understanding germline specification in plants could be advantageous for agricultural applications. In recent decades, substantial efforts have been made to understand germline specification in several plant species, including Arabidopsis, rice, and maize. However, our knowledge of germline specification in many agronomically important plant species remains obscure. Here, we characterized the female germline specification and subsequent female gametophyte development in pineapple using callose staining, cytological, and whole-mount immunolocalization analyses. We also determined the male germline specification and gametophyte developmental timeline and observed male meiotic behavior using chromosome spreading assays. Furthermore, we identified 229 genes that are preferentially expressed at the megaspore mother cell (MMC) stage during ovule development and 478 genes that are preferentially expressed at the pollen mother cell (PMC) stage of anther development using comparative transcriptomic analysis. The biological functions, associated regulatory pathways and expression patterns of these genes were also analyzed. Our study provides a convenient cytological reference for exploring pineapple germline development and a molecular basis for the future functional analysis of germline specification in related plant species.

Flower evolution in the presence of heterospecific gene flow and its contribution to lineage divergence.

The success of species depends on their ability to exploit ecological resources in order to optimize their reproduction. However, species are not usually found within single-species ecosystems but in complex communities. Because of their genetic relatedness, closely related lineages tend to cluster within the same ecosystem, rely on the same resources, and be phenotypically similar. In sympatry, they will therefore compete for the same resources and, in the case of flowering plants, exchange their genes through heterospecific pollen transfer. These interactions, nevertheless, pose significant challenges to species co-existence because they can lead to resource limitation and reproductive interference. In such cases, divergent selective pressures on floral traits will favour genotypes that isolate or desynchronize the reproduction of sympatric lineages. The resulting displacement of reproductive characters will, in turn, lead to pre-mating isolation and promote intraspecific divergence, thus initiating or reinforcing the speciation process. In this review, we discuss the current theoretical and empirical knowledge on the influence of heterospecific pollen transfer on flower evolution, highlighting its potential to uncover the ecological and genomic constraints shaping the speciation process.

A Similar Genetic Architecture Underlies the Convergent Evolution of the Selfing Syndrome in Capsella.

Whether, and to what extent, phenotypic evolution follows predictable genetic paths remains an important question in evolutionary biology. Convergent evolution of similar characters provides a unique opportunity to address this question. The transition to selfing and the associated changes in flower morphology are among the most prominent examples of repeated evolution in plants. In this study, we take advantage of the independent transitions to self-fertilization in the genus Capsella to compare the similarities between parallel modifications of floral traits and test for genetic and developmental constraints imposed on flower evolution in the context of the selfing syndrome. Capsella rubella and Capsella orientalis emerged independently but evolved almost identical flower characters. Not only is the evolutionary outcome identical but the same developmental strategies underlie the convergent reduction of flower size. This has been associated with convergent evolution of gene expression changes. The transcriptomic changes common to both selfing lineages are enriched in genes with low network connectivity and with organ-specific expression patterns. Comparative genetic mapping also suggests that, at least in the case of petal size evolution, these similarities have a similar genetic basis. Based on these results, we hypothesize that the limited availability of low-pleiotropy paths predetermines closely related species to similar evolutionary outcomes.

Retracing the molecular basis and evolutionary history of the loss of benzaldehyde emission in the genus Capsella.

The transition from pollinator-mediated outbreeding to selfing has occurred many times in angiosperms. This is generally accompanied by a reduction in traits attracting pollinators, including reduced emission of floral scent. In Capsella, emission of benzaldehyde as a main component of floral scent has been lost in selfing C. rubella by mutation of cinnamate-CoA ligase CNL1. However, the biochemical basis and evolutionary history of this loss remain unknown, as does the reason for the absence of benzaldehyde emission in the independently derived selfer Capsella orientalis. We used plant transformation, in vitro enzyme assays, population genetics and quantitative genetics to address these questions. CNL1 has been inactivated twice independently by point mutations in C. rubella, causing a loss of enzymatic activity. Both inactive haplotypes are found within and outside of Greece, the centre of origin of C. rubella, indicating that they arose before its geographical spread. By contrast, the loss of benzaldehyde emission in C. orientalis is not due to an inactivating mutation in CNL1. CNL1 represents a hotspot for mutations that eliminate benzaldehyde emission, potentially reflecting the limited pleiotropy and large effect of its inactivation. Nevertheless, even closely related species have followed different evolutionary routes in reducing floral scent.

A high-throughput amplicon-based method for estimating outcrossing rates.

BACKGROUND: The outcrossing rate is a key determinant of the population-genetic structure of species and their long-term evolutionary trajectories. However, determining the outcrossing rate using current methods based on PCR-genotyping individual offspring of focal plants for multiple polymorphic markers is laborious and time-consuming. RESULTS: We have developed an amplicon-based, high-throughput enabled method for estimating the outcrossing rate and have applied this to an example of scented versus non-scented Capsella (Shepherd's Purse) genotypes. Our results show that the method is able to robustly capture differences in outcrossing rates. They also highlight potential biases in the estimates resulting from differential haplotype sharing of the focal plants with the pollen-donor population at individual amplicons. CONCLUSIONS: This novel method for estimating outcrossing rates will allow determining this key population-genetic parameter with high-throughput across many genotypes in a population, enabling studies into the genetic determinants of successful pollinator attraction and outcrossing.

Mechanisms Underlying the Environmentally Induced Plasticity of Leaf Morphology.

The primary function of leaves is to provide an interface between plants and their environment for gas exchange, light exposure and thermoregulation. Leaves have, therefore a central contribution to plant fitness by allowing an efficient absorption of sunlight energy through photosynthesis to ensure an optimal growth. Their final geometry will result from a balance between the need to maximize energy uptake while minimizing the damage caused by environmental stresses. This intimate relationship between leaf and its surroundings has led to an enormous diversification in leaf forms. Leaf shape varies between species, populations, individuals or even within identical genotypes when those are subjected to different environmental conditions. For instance, the extent of leaf margin dissection has, for long, been found to inversely correlate with the mean annual temperature, such that Paleobotanists have used models based on leaf shape to predict the paleoclimate from fossil flora. Leaf growth is not only dependent on temperature but is also regulated by many other environmental factors such as light quality and intensity or ambient humidity. This raises the question of how the different signals can be integrated at the molecular level and converted into clear developmental decisions. Several recent studies have started to shed the light on the molecular mechanisms that connect the environmental sensing with organ-growth and patterning. In this review, we discuss the current knowledge on the influence of different environmental signals on leaf size and shape, their integration as well as their importance for plant adaptation.

Capsella.

Capsella species, including the well-known Shephard's purse, are characterized by small white flowers and heart-shaped fruit. But read on to discover the fascinating insights these diminutive plants have to reveal about the evolution of self-fertilizing plants.

Successive duplication-divergence mechanisms at the RCO locus contributed to leaf shape diversity in the Brassicaceae.

Gene duplication is a major driver for the increase of biological complexity. The divergence of newly duplicated paralogs may allow novel functions to evolve, while maintaining the ancestral one. Alternatively, partitioning the ancestral function among paralogs may allow parts of that role to follow independent evolutionary trajectories. We studied the REDUCED COMPLEXITY (RCO) locus, which contains three paralogs that have evolved through two independent events of gene duplication, and which underlies repeated events of leaf shape evolution within the Brassicaceae. In particular, we took advantage of the presence of three potentially functional paralogs in Capsella to investigate the extent of functional divergence among them. We demonstrate that the RCO copies control growth in different areas of the leaf. Consequently, the copies that are retained active in the different Brassicaceae lineages contribute to define the leaf dissection pattern. Our results further illustrate how successive gene duplication events and subsequent functional divergence can increase trait evolvability by providing independent evolutionary trajectories to specialized functions that have an additive effect on a given trait.

At-MINI ZINC FINGER2 and Sl-INHIBITOR OF MERISTEM ACTIVITY, a Conserved Missing Link in the Regulation of Floral Meristem Termination in Arabidopsis and Tomato.

In angiosperms, the gynoecium is the last structure to develop within the flower due to the determinate fate of floral meristem (FM) stem cells. The maintenance of stem cell activity before its arrest at the stage called FM termination affects the number of carpels that develop. The necessary inhibition at this stage of WUSCHEL (WUS), which is responsible for stem cell maintenance, involves a two-step mechanism. Direct repression mediated by the MADS domain transcription factor AGAMOUS (AG), followed by indirect repression requiring the C2H2 zinc-finger protein KNUCKLES (KNU), allow for the complete termination of floral stem cell activity. Here, we show that Arabidopsis thaliana MINI ZINC FINGER2 (AtMIF2) and its homolog in tomato (Solanum lycopersicum), INHIBITOR OF MERISTEM ACTIVITY (SlIMA), participate in the FM termination process by functioning as adaptor proteins. AtMIF2 and SlIMA recruit AtKNU and SlKNU, respectively, to form a transcriptional repressor complex together with TOPLESS and HISTONE DEACETYLASE19. AtMIF2 and SlIMA bind to the WUS and SlWUS loci in the respective plants, leading to their repression. These results provide important insights into the molecular mechanisms governing (FM) termination and highlight the essential role of AtMIF2/SlIMA during this developmental step, which determines carpel number and therefore fruit size.

Evolvability of flower geometry: Convergence in pollinator-driven morphological evolution of flowers.

Flowers represent a key innovation during plant evolution. Driven by reproductive optimization, evolution of flower morphology has been central in boosting species diversification. In most cases, this has happened through specialized interactions with animal pollinators and subsequent reduction of gene flow between specialized morphs. While radiation has led to an enormous variability in flower forms and sizes, recurrent evolutionary patterns can be observed. Here, we discuss the targets of selection involved in major trends of pollinator-driven flower evolution. We review recent findings on their adaptive values, developmental grounds and genetic bases, in an attempt to better understand the repeated nature of pollinator-driven flower evolution. This analysis highlights how structural innovation can provide flexibility in phenotypic evolution, adaptation and speciation.

Standing genetic variation in a tissue-specific enhancer underlies selfing-syndrome evolution in Capsella.

Mating system shifts recurrently drive specific changes in organ dimensions. The shift in mating system from out-breeding to selfing is one of the most frequent evolutionary transitions in flowering plants and is often associated with an organ-specific reduction in flower size. However, the evolutionary paths along which polygenic traits, such as size, evolve are poorly understood. In particular, it is unclear how natural selection can specifically modulate the size of one organ despite the pleiotropic action of most known growth regulators. Here, we demonstrate that allelic variation in the intron of a general growth regulator contributed to the specific reduction of petal size after the transition to selfing in the genus Capsella Variation within this intron affects an organ-specific enhancer that regulates the level of STERILE APETALA (SAP) protein in the developing petals. The resulting decrease in SAP activity leads to a shortening of the cell proliferation period and reduced number of petal cells. The absence of private polymorphisms at the causal region in the selfing species suggests that the small-petal allele was captured from standing genetic variation in the ancestral out-crossing population. Petal-size variation in the current out-crossing population indicates that several small-effect mutations have contributed to reduce petal-size. These data demonstrate how tissue-specific regulatory elements in pleiotropic genes contribute to organ-specific evolution. In addition, they provide a plausible evolutionary explanation for the rapid evolution of flower size after the out-breeding-to-selfing transition based on additive effects of segregating alleles.

Fruit shape diversity in the Brassicaceae is generated by varying patterns of anisotropy.

Fruits exhibit a vast array of different 3D shapes, from simple spheres and cylinders to more complex curved forms; however, the mechanism by which growth is oriented and coordinated to generate this diversity of forms is unclear. Here, we compare the growth patterns and orientations for two very different fruit shapes in the Brassicaceae: the heart-shaped Capsella rubella silicle and the near-cylindrical Arabidopsis thaliana silique. We show, through a combination of clonal and morphological analyses, that the different shapes involve different patterns of anisotropic growth during three phases. These experimental data can be accounted for by a tissue-level model in which specified growth rates vary in space and time and are oriented by a proximodistal polarity field. The resulting tissue conflicts lead to deformation of the tissue as it grows. The model allows us to identify tissue-specific and temporally specific activities required to obtain the individual shapes. One such activity may be provided by the valve-identity gene FRUITFULL, which we show through comparative mutant analysis to modulate fruit shape during post-fertilisation growth of both species. Simple modulations of the model presented here can also broadly account for the variety of shapes in other Brassicaceae species, thus providing a simplified framework for fruit development and shape diversity.