Maria Cartolano

A conserved microRNA module exerts homeotic control over Petunia hybrida and Antirrhinum majus floral organ identity

It is commonly thought that deep phylogenetic conservation of plant microRNAs (miRNAs) and their targets indicates conserved regulatory functions. We show that the blind (bl) mutant of Petunia hybrida and the fistulata (fis) mutant of Antirrhinum majus, which have similar homeotic phenotypes, are recessive alleles of two homologous miRNA-encoding genes. The BL and FIS genes control the spatial restriction of homeotic class C genes to the inner floral whorls, but their ubiquitous early floral expression patterns are in contradiction with a potential role in patterning C gene expression. We provide genetic evidence for the unexpected function of the MIRFIS and MIRBL genes in the center of the flower and propose a dynamic mechanism underlying their regulatory role. Notably, Arabidopsis thaliana, a more distantly related species, also contains this miRNA module but does not seem to use it to confine early C gene expression to the center of the flower.

Arabidopsis thaliana Leaf Form Evolved via Loss of KNOX Expression in Leaves in Association with a Selective Sweep

Morphological diversity is often caused by altered gene expression of key developmental regulators. However, the precise developmental trajectories through which morphologies evolved remain poorly understood. It is also unclear to what degree genetic changes contributing to morphological divergence were fixed by natural selection. Here we investigate these problems in the context of evolutionary developmental transitions that produced the simple unlobed leaf of the model species Arabidopsis thaliana. We demonstrate that A. thaliana leaf shape likely derived from a more complex lobed ancestral state that persists in extant Arabidopsis species. We also show that evolution of the unlobed leaf form in A. thaliana involved loss of expression of the knotted1-like homeobox gene SHOOTMERISTEMLESS (STM) in leaves and that cis-regulatory divergence contributed to this process. Further, we provide evidence for a selective sweep at the A. thaliana STM locus, indicating that loss of STM expression in A. thaliana leaves may have been fixed by positive selection. In summary, our data provide key information as to when and how the characteristic leaf form of A. thaliana evolved.

Flower development: the antirrhinum perspective

Abstract Research with the snapdragon, Antirrhinum majus, has a long history with many highlights, making this species a significant model system for comparative genetic, molecular, and ecological studies. In this chapter, we focus interest on flower development, in particular the genetic control of floral organ identity, floral asymmetry, and petal cell‐type specification, where results obtained with Antirrhinum provided the first insights into the underlying molecular mechanisms, leading to advances in the field. In addition to reviewing past and recent scientific achievements, we propose simple models to aid the understanding of complex genetic observations and also to resolve the inconsistencies and contradictions, which inhibit the general application of existing models to other species. In particular, we propose a revision of the ABC‐model to reflect better the experimental results obtained in a variety of model species including Arabidopsis. For this we revive the (A)BC‐scheme in which sepal identity follows from floral meristem identity and represents the ground state for floral organs. In addition to controlling sepal identity, the complex (A)‐function performs several roles that are necessary for the initiation, maintenance, spatial restriction, and functionality of the B‐ and C‐organ identity functions. By providing information on current resources for molecular research and newly arising research areas we intend to encourage the scientific community to utilize Antirrhinum for research in the future.

Heterochrony underpins natural variation in Cardamine hirsuta leaf form

Significance A key problem in biology is whether the same processes underlie morphological variation between and within species. Here, we show that the causes of leaf shape diversity at these two evolutionary scales can be divergent. Some species have simple leaves, whereas others bear complex leaves comprising leaflets. Previous work indicated that these interspecific differences result mostly from variation in local tissue growth and patterning. Now we find that a different process, age-dependent shape progression, underlies within-species variation in complex leaf morphology. Specifically, in plants with accelerated aging and early flowering, leaves progress to adult shapes with more leaflets, faster than their slower-aging counterparts. This mechanism coordinates leaf development with reproductive timing and may influence resource allocation to seeds. A key problem in biology is whether the same processes underlie morphological variation between and within species. Here, by using plant leaves as an example, we show that the causes of diversity at these two evolutionary scales can be divergent. Some species like the model plant Arabidopsis thaliana have simple leaves, whereas others like the A. thaliana relative Cardamine hirsuta bear complex leaves comprising leaflets. Previous work has shown that these interspecific differences result mostly from variation in local tissue growth and patterning. Now, by cloning and characterizing a quantitative trait locus (QTL) for C. hirsuta leaf shape, we find that a different process, age-dependent progression of leaf form, underlies variation in this trait within species. This QTL effect is caused by cis-regulatory variation in the floral repressor ChFLC, such that genotypes with low-expressing ChFLC alleles show both early flowering and accelerated age-dependent changes in leaf form, including faster leaflet production. We provide evidence that this mechanism coordinates leaf development with reproductive timing and may help to optimize resource allocation to the next generation.

The Cardamine hirsuta genome offers insight into the evolution of morphological diversity

Finding causal relationships between genotypic and phenotypic variation is a key focus of evolutionary biology, human genetics and plant breeding. To identify genome-wide patterns underlying trait diversity, we assembled a high-quality reference genome of Cardamine hirsuta, a close relative of the model plant Arabidopsis thaliana. We combined comparative genome and transcriptome analyses with the experimental tools available in C. hirsuta to investigate gene function and phenotypic diversification. Our findings highlight the prevalent role of transcription factors and tandem gene duplications in morphological evolution. We identified a specific role for the transcriptional regulators PLETHORA5/7 in shaping leaf diversity and link tandem gene duplication with differential gene expression in the explosive seed pod of C. hirsuta. Our work highlights the value of comparative approaches in genetically tractable species to understand the genetic basis for evolutionary change.

Enhanced AGAMOUS expression in the centre of the Arabidopsis flower causes ectopic expression over its outer expression boundaries

Spatial regulation of C-function genes controlling reproductive organ identity in the centre of the flower can be achieved by adjusting the level of their expression within the genuine central expression domain in Antirrhinum and Petunia. Loss of this control in mutants is revealed by enhanced C-gene expression in the centre and by lateral expansion of the C-domain. In order to test whether the level of central C-gene expression and hence the principle of ‘regulation by tuning’ also applies to spatial regulation of the C-function gene AGAMOUS (AG) in Arabidopsis, we generated transgenic plants with enhanced central AG expression by using stem cell-specific CLAVATA3 (CLV3) regulatory sequences to drive transcription of the AG cDNA. The youngest terminal flowers on inflorescences of CLV3::AG plants displayed homeotic features in their outer whorls indicating ectopic AG expression. Dependence of the homeotic feature on the age of the plant is attributed to the known overall weakening of repressive mechanisms controlling AG. Monitoring AG with an AG-I::GUS reporter construct suggests ectopic AG expression in CLV3::AG flowers when AG in the inflorescence is still repressed, although in terminating inflorescence meristems, AG expression expands to all tissues. Supported by genetic tests, we conclude that upon enhanced central AG expression, the C-domain laterally expands necessitating tuning of the expression level of C-function genes in the wild type. The tuning mechanism in C-gene regulation in Arabidopsis is discussed as a late security switch that ensures wild-type C-domain control when other repressive mechanism starts to fade and fail.

cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing.

The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.

Invasion history of Cardamine hirsuta in Japan inferred from genetic analyses of herbarium specimens and current populations

Multiple introductions of a species are thought to enhance its invasion success by increasing genotypic diversity; this involves frequent crossing among different lineages. However, genetic diversity through crossing is less likely in autogamous species. To understand the impact of multiple introductions on the colonization success of autogamous species, we studied hairy bittercress, Cardamine hirsuta, which invaded Japan several decades ago. We detected temporal changes in its population structure using nine microsatellite markers amplified from leaf samples collected from 87 sites between 2009 and 2010, and herbarium specimens collected between 1988 and 2007. To examine whether the phenotypic variation corresponded with the genetic population structure, we also investigated the geographic variation in the lateral stamen number of this species across 49 sites. The present populations can be divided into three genetic groups, which are distributed in northern, eastern, and western Japan. This finding suggests that there are three invasive lineages (North, East, and West) in Japan. The geographic variation in lateral stamen number corresponded to the distributions of these lineages. The former distributions of the North and West lineages mostly corresponded to those found at present, but they were also historically found in eastern Japan. However, the East lineage has apparently expanded into eastern Japan, resulting in a change in dominant lineages over only a few decades. For the autogamous C. hirsuta, multiple introductions contributed toward colonization success over a wider range, which was associated with a local change in the dominant lineages.

Pleiotropic effect of the Flowering Locus C on plant resistance and defence against insect herbivores

Summary 1. Plants vary widely in the extent to which they defend themselves against herbivores. Because the resources available to plants are often site-specific, variation among sites dictates investment into defence, and may reveal a growth-defence trade-off. Moreover, plants that have evolved different life-history strategies in different environments may situate themselves on this trade-off curve differently. For instance, plants that flower later have a longer vegetative lifespan, and may accordingly defend themselves differently than those that flower earlier. 2. Here, we tested whether late-flowering plants, with a longer vegetative lifespan, invest more in defence than early-flowering plants, using recombinant genotypes of the annual herb Cardamine hirsuta that differ in flowering time as a result of differences in the activity of the major floral repressor Flowering Locus C (FLC). 3. We found that variation at FLC was mainly responsible for regulating flowering time and allocation to reproduction, but this partially depended on where the plants grew. We also found that variation at FLC mediated plant allocation to defence, with late- flowering plants producing higher levels of total glucosinolates and stress-related phytohormones. Nonetheless, plant growth and the qualitative values of plant defence and plant resistance against specialist herbivores were mainly independent from FLC. 4. Synthesis - Our results highlight pleiotropic effects associated with flowering-time genes that might influence plant defence and plant-herbivore interactions.