Custom methods to identify conserved genetic modules applied to novel transcriptomic data from Amborella trichopoda.

We have devised a procedure for the inter-species comparison of transcriptomic data and used this procedure to reconstruct the expression dynamics of major genetic modules that were present at least 149 million years ago in the most recent common ancestor of living angiosperms. We began by using laser-assisted microdissection to generate novel transcriptomic data from female flower tissues of Amborella trichopoda, the likely sister to all other living angiosperms. We then employed a gene-expression clustering method, followed by a custom procedure to compare genetic modules on the basis of gene orthology between Amborella and the molecular-genetic model angiosperm Arabidopsis thaliana. Using this protocol, we succeeded in identifying nine major genetic modules that appear to have conserved their expression dynamics from an early stage in angiosperm evolution. The genes of these modules, representing over 5000 orthogroups, include around one third of those known to control female reproductive development in Arabidopsis. Our study constitutes a proof of concept for the comparison of transcriptomic data between widely diverged plant species and represents a first step in the large-scale analysis of gene expression dynamics in a macro-evolutionary context.

A derived ZW chromosome system in Amborella trichopoda, representing the sister lineage to all other extant flowering plants.

The genetic basis and evolution of sex determination in dioecious plants is emerging as an active area of research with exciting advances in genome sequencing and analysis technologies. As the sole species within the sister lineage to all other extant flowering plants, Amborella trichopoda is an important model for understanding the evolution and development of flowers. Plants typically produce only male or female flowers, but sex determination mechanisms are unknown for the species. Sequence data derived from plants of natural origin and an F1 mapping population were used to identify sex-linked genes and the nonrecombining region. Amborella trichopoda has a ZW sex determination system. Analysis of genes in a 4 Mb nonrecombining sex-determination region reveals recent divergence of Z and W gametologs, and few Z- and W-specific genes. The sex chromosomes of A. trichopoda evolved less than 16.5 Myr ago, long after the divergence of the extant angiosperms.

Characterization of the Bubblegum acyl-CoA synthetase of Microchloropsis gaditana.

The metabolic pathways of glycerolipids are well described in cells containing chloroplasts limited by a two-membrane envelope but not in cells containing plastids limited by four membranes, including heterokonts. Fatty acids (FAs) produced in the plastid, palmitic and palmitoleic acids (16:0 and 16:1), are used in the cytosol for the synthesis of glycerolipids via various routes, requiring multiple acyl-Coenzyme A (CoA) synthetases (ACS). Here, we characterized an ACS of the Bubblegum subfamily in the photosynthetic eukaryote Microchloropsis gaditana, an oleaginous heterokont used for the production of lipids for multiple applications. Genome engineering with TALE-N allowed the generation of MgACSBG point mutations, but no knockout was obtained. Point mutations triggered an overall decrease of 16:1 in lipids, a specific increase of unsaturated 18-carbon acyls in phosphatidylcholine and decrease of 20-carbon acyls in the betaine lipid diacylglyceryl-trimethyl-homoserine. The profile of acyl-CoAs highlighted a decrease in 16:1-CoA and 18:3-CoA. Structural modeling supported that mutations affect accessibility of FA to the MgACSBG reaction site. Expression in yeast defective in acyl-CoA biosynthesis further confirmed that point mutations affect ACSBG activity. Altogether, this study supports a critical role of heterokont MgACSBG in the production of 16:1-CoA and 18:3-CoA. In M. gaditana mutants, the excess saturated and monounsaturated FAs were diverted to triacylglycerol, thus suggesting strategies to improve the oil content in this microalga.

Immediate targets of ETTIN suggest a key role for pectin methylesterase inhibitors in the control of Arabidopsis gynecium development.

The control of gynecium development in Arabidopsis thaliana by the auxin response factor ETTIN (ETT) correlates with a reduction in the methylesterification of cell-wall pectins and a decrease in cell-wall stiffness in the valve tissues of the ovary. Here, we determine the list of genes rapidly regulated following the in-vivo activation of an ETT fusion protein, and show these to be significantly enriched in genes encoding cell-wall proteins, including several pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs). We also perform a genome-wide scan for potential ETT-binding sites, and incorporate the results of this procedure into a comparison of datasets, derived using four distinct methods, to identify genes regulated directly or indirectly by ETT. We conclude from our combined analyses that PMEIs are likely to be key actors that mediate the regulation of gynecium development by ETT, while ETT may simultaneously regulate PMEs to prevent exaggerated developmental effects from the regulation of PMEIs. We also postulate the existence of one or more rapidly-acting intermediate factors in the transcriptional regulation of PMEs and PMEIs by ETT.

Evidence for the Extensive Conservation of Mechanisms of Ovule Integument Development Since the Most Recent Common Ancestor of Living Angiosperms.

The ovules and seeds of most angiosperm groups are enclosed by two integuments, whose evolutionary origins are considerably separated in time, as the inner integument arose over 300 million years ago (MYA) in an ancestor of all living seed plants, while the outer integument arose, perhaps as recently as 164 MYA, in an ancestor of all living angiosperms. Studies of the model angiosperm Arabidopsis thaliana indicate that the mechanisms of development of the inner and outer integuments depend on largely different sets of molecular players. However, it was not known, in most cases, whether these differences were already present in early flowering plants, or arose later in the Arabidopsis lineage. Here, we analyze the expression patterns of integument regulators in Amborella trichopoda, the likely sister to all other living angiosperms. The data obtained indicate that regulators of the YABBY, KANADI, and homeodomain-leucine zipper class III transcription factor families have largely conserved their integument-specific expression profiles in the Amborella and Arabidopsis lineages since the most recent common ancestor (MRCA) of living angiosperms. We identified only one case, involving the paralogous genes ETTIN and AUXIN RESPONSE FACTOR4, in which integument-specific expression patterns had clearly diverged between Amborella and Arabidopsis. We use the data obtained to partially reconstruct molecular mechanisms of integument development in the MRCA of living angiosperms and discuss our findings in the context of alternative hypotheses for the origin of the angiosperm outer integument.

Evidence for the Regulation of Gynoecium Morphogenesis by ETTIN via Cell Wall Dynamics.

ETTIN (ETT) is an atypical member of the AUXIN RESPONSE FACTOR family of transcription factors that plays a crucial role in tissue patterning in the Arabidopsis (Arabidopsis thaliana) gynoecium. Though recent insights have provided valuable information on ETT's interactions with other components of auxin signaling, the biophysical mechanisms linking ETT to its ultimate effects on gynoecium morphology were until now unknown. Here, using techniques to assess cell-wall dynamics during gynoecium growth and development, we provide a coherent body of evidence to support a model in which ETT controls the elongation of the valve tissues of the gynoecium through the positive regulation of pectin methylesterase (PME) activity in the cell wall. This increase in PME activity results in an increase in the level of demethylesterified pectins and a consequent reduction in cell wall stiffness, leading to elongation of the valves. Though similar biophysical mechanisms have been shown to act in the stem apical meristem, leading to the expansion of organ primordia, our findings demonstrate that regulation of cell wall stiffness through the covalent modification of pectin also contributes to tissue patterning within a developing plant organ.

Transcriptional induction of cell wall remodelling genes is coupled to microtubule-driven growth isotropy at the shoot apex in Arabidopsis.

The shoot apical meristem of higher plants continuously generates new tissues and organs through complex changes in growth rates and directions of its individual cells. Cell growth, which is driven by turgor pressure, largely depends on the cell walls, which allow cell expansion through synthesis and structural changes. A previous study revealed a major contribution of wall isotropy in organ emergence, through the disorganization of cortical microtubules. We show here that this disorganization is coupled with the transcriptional control of genes involved in wall remodelling. Some of these genes are induced when microtubules are disorganized and cells shift to isotropic growth. Mechanical modelling shows that this coupling has the potential to compensate for reduced cell expansion rates induced by the shift to isotropic growth. Reciprocally, cell wall loosening induced by different treatments or altered cell wall composition promotes a disruption of microtubule alignment. Our data thus indicate the existence of a regulatory module activated during organ outgrowth, linking microtubule arrangements to cell wall remodelling.

The analysis of Gene Regulatory Networks in plant evo-devo.

We provide an overview of methods and workflows that can be used to investigate the topologies of Gene Regulatory Networks (GRNs) in the context of plant evolutionary-developmental (evo-devo) biology. Many of the species that occupy key positions in plant phylogeny are poorly adapted as laboratory models and so we focus here on techniques that can be efficiently applied to both model and non-model species of interest to plant evo-devo. We outline methods that can be used to describe gene expression patterns and also to elucidate the transcriptional, post-transcriptional, and epigenetic regulatory mechanisms underlying these patterns, in any plant species with a sequenced genome. We furthermore describe how the technique of Protein Resurrection can be used to confirm inferences on ancestral GRNs and also to provide otherwise-inaccessible points of reference in evolutionary histories by exploiting paralogues generated in gene and whole genome duplication events. Finally, we argue for the better integration of molecular data with information from paleobotanical, paleoecological, and paleogeographical studies to provide the fullest possible picture of the processes that have shaped the evolution of plant development.