Amanda L. Rychel

Out of the Mouths of Plants: The Molecular Basis of the Evolution and Diversity of Stomatal Development

Stomata are microscopic valves on the plant epidermis that played a critical role in the evolution of land plants. Studies in the model dicot Arabidopsis thaliana have identified key transcription factors and signaling pathways controlling stomatal patterning and differentiation. Three paralogous Arabidopsis basic helix-loop-helix proteins, SPEECHLESS (SPCH), MUTE, and FAMA, mediate sequential steps of cell-state transitions together with their heterodimeric partners SCREAM (SCRM) and SCRM2. Cell–cell signaling components, including putative ligands, putative receptors, and mitogen-activated protein kinase cascades, orient asymmetric cell divisions and prevent overproduction and clustering of stomata. The recent availability of genome sequence and reverse genetics tools for model monocots and basal land plants allows for the examination of the conservation of genes important in stomatal patterning and differentiation. Studies in grasses have revealed that divergence of SPCH-MUTE-FAMA predates the evolutionary split of monocots and dicots and that these proteins show conserved and novel roles in stomatal differentiation. By contrast, specific asymmetric cell divisions in Arabidopsis and grasses require unique molecular components. Molecular phylogenetic analysis implies potential conservation of signaling pathways and prototypical functions of the transcription factors specifying stomatal differentiation.

Plant twitter: ligands under 140 amino acids enforcing stomatal patterning

Stomata are an essential land plant innovation whose patterning and density are under genetic and environmental control. Recently, several putative ligands have been discovered that influence stomatal density, and they all belong to the EPIDERMAL PATTERNING FACTOR-LIKE family of secreted cysteine-rich peptides. Two of these putative ligands, EPF1 and EPF2, are expressed exclusively in the stomatal lineage cells and negatively regulate stomatal density. A third, EPFL6 or CHALLAH, is also a negative regulator of density, but is expressed subepidermally in the hypocotyl. A fourth, EPFL9 or STOMAGEN, is expressed in the mesophyll tissues and is a positive regulator of density. Genetic evidence suggests that these ligands may compete for the same receptor complex. Proper stomatal patterning is likely to be an intricate process involving ligand competition, regional specificity, and communication between tissue layers. EPFL-family genes exist in the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii, and rice, Oryza sativa, and their sequence analysis yields several genes some of which are related to EPF1, EPF2, EPFL6, and EPFL9. Presence of these EPFL family members in the basal land plants suggests an exciting hypothesis that the genetic components for stomatal patterning originated early in land plant evolution.

Molecular phylogeny of hemichordata, with updated status of deep-sea enteropneusts.

Hemichordates have occupied a central role in hypotheses of deuterostome and early chordate evolution. However, surprisingly little is understood about evolution within hemichordates, including hemichordate ancestral characters that may relate to other deuterostome taxa. Previous phylogenetic studies suggested that enteropneust worms are either monophyletic (based on 28S rDNA) or paraphyletic (based on 18S rDNA). Here, we expand the number of hemichordate taxa used in phylogenetic analyses for 18S rDNA data and employ more quickly evolving mitochondrial gene sequences. Novel data from an undescribed deep-sea enteropneust species similar to Torquarator bullocki and a Gulf Stream tornaria larva suggest that these taxa are closely allied to or possibly within Ptychoderidae. Saxipendium coronatum, another deep-sea species commonly called the spaghetti worm, is shown to be a member of Harrimaniidae. Recognition of these deep-sea lineages as distinct families calls into question features used in hemichordate taxonomy. In the new analyses, enteropneusts fall into two distinct monophyletic clades, with the colonial pterobranchs sister to Harrimaniidae, similar to earlier published 18S results. These results indicate that colonial pterobranchs may have evolved from a solitary acorn worm-like hemichordate ancestor. If true, pterobranchs would be unlikely to represent the deuterostome ancestral form as has been suggested by many traditional theories of deuterostome evolution.

Molecular Framework of a Regulatory Circuit Initiating Two-Dimensional Spatial Patterning of Stomatal Lineage

Stomata, valves on the plant epidermis, are critical for plant growth and survival, and the presence of stomata impacts the global water and carbon cycle. Although transcription factors and cell-cell signaling components regulating stomatal development have been identified, it remains unclear as to how their regulatory interactions are translated into two-dimensional patterns of stomatal initial cells. Using molecular genetics, imaging, and mathematical simulation, we report a regulatory circuit that initiates the stomatal cell-lineage. The circuit includes a positive feedback loop constituting self-activation of SCREAMs that requires SPEECHLESS. This transcription factor module directly binds to the promoters and activates a secreted signal, EPIDERMAL PATTERNING FACTOR2, and the receptor modifier TOO MANY MOUTHS, while the receptor ERECTA lies outside of this module. This in turn inhibits SPCH, and hence SCRMs, thus constituting a negative feedback loop. Our mathematical model accurately predicts all known stomatal phenotypes with the inclusion of two additional components to the circuit: an EPF2-independent negative-feedback loop and a signal that lies outside of the SPCH•SCRM module. Our work reveals the intricate molecular framework governing self-organizing two-dimensional patterning in the plant epidermis.

Anterior regeneration in the hemichordate Ptychodera flava.

Ptychodera flava is a hemichordate whose anterior structures regenerate reproducibly from posterior trunk pieces when amputated. We characterized the cellular processes of anterior regeneration with respect to programmed cell death and cell proliferation, after wound healing. We found scattered proliferating cells at day 2 of regeneration using a proliferating cell nuclear antigen antibody. On day 4, most proliferating cells were associated with the nerve tract under the epidermis, and on day 6, a small proboscis derived from proliferated cells was regenerated, and a mouth had broken though the epidermis. TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end‐labeling) detected elevated levels of apoptosis in the endoderm that began furthest away from the region of wound healing, then moved anteriorly over 8 days. Posterior to anterior apoptosis is likely to remove digestive endoderm for later differentiation of pharyngeal endoderm. We hypothesize that P. flava regeneration is nerve dependent and that remodeling in the gut endoderm plays an important role in regeneration. Developmental Dynamics 237:3222–3232, 2008.

Regeneration in Hemichordates and Echinoderms

Hemichordates are closely related to echinoderms, which are remarkable for their powers of regeneration. Among hemichordates, some enteropneust worms show dramatic regeneration, though this process has not been as well studied as in echinoderms. Unlike echinoderms, which are pentameral in adult form, hemichordates exhibit bilateral symmetry throughout the life cycle. Adult body regeneration in hemichordates may therefore show similar molecular patterning to chordate regeneration. In this chapter, we review the original literature about regeneration in hemichordates. We present our results from Glossobalanus berkeleyi and Ptychodera flava, the latter of which reliably regenerates anterior structures in the laboratory. When P. flava is bisected, the wound at the anterior end of the posterior half heals, followed by outgrowth of a blastema that becomes the new proboscis. After the proboscis develops, the mouth opens, the collar folds up, and gill slits are formed by tissue remodeling in the posterior worm fragment. Renewed interest in hemichordate regeneration, combined with the fact that it can be elicited in the lab, sets the stage for using molecular markers for stem cells and differentiated tissue in order to characterize the cellular and molecular events occurring during hemichordate regeneration. Hemichordate data presented here suggests that the ability to regenerate using stem cells may be common in ambulacrarian deuterostomes, the sister group to chordates. Understanding the molecular basis of regeneration in hemichordates may lead the way to methods for stimulating regeneration in vertebrates, including in humans for therapeutic purposes.

An ensemble of structures of Burkholderia pseudomallei 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase.

An ensemble of crystal structures are reported for 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase from B. pseudomallei. The structures include two vanadate complexes, revealing the structure of a close analogue of the transition state for phosphate transfer.

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

The first crystal structure is reported of a glutaryl-CoA dehydrogenase in the apo state without flavin adenine dinucleotide cofactor bound. Additional structures with small molecules complexed in the catalytic active site were obtained by fragment-based screening.