Low-Speed Clinorotation of Brachypodium distachyon and Arabidopsis thaliana Seedlings Triggers Root Tip Curvatures That Are Reminiscent of Gravitropism.

Clinostats are instruments that continuously rotate biological specimens along an axis, thereby averaging their orientation relative to gravity over time. Our previous experiments indicated that low-speed clinorotation may itself trigger directional root tip curvature. In this project, we have investigated the root curvature response to low-speed clinorotation using Arabidopsis thaliana and Brachypodium distachyon seedlings as models. We show that low-speed clinorotation triggers root tip curvature in which direction is dictated by gravitropism during the first half-turn of clinorotation. We also show that the angle of root tip curvature is modulated by the speed of clinorotation. Arabidopsis mutations affecting gravity susception (pgm) or gravity signal transduction (arg1, toc132) are shown to affect the root tip curvature response to low-speed clinorotation. Furthermore, low-speed vertical clinorotation triggers relocalization of the PIN3 auxin efflux facilitator to the lateral membrane of Arabidopsis root cap statocytes, and creates a lateral gradient of auxin across the root tip. Together, these observations support a role for gravitropism in modulating root curvature responses to clinorotation. Interestingly, distinct Brachypodium distachyon accessions display different abilities to develop root tip curvature responses to low-speed vertical clinorotation, suggesting the possibility of using genome-wide association studies to further investigate this process.

Gravity sensing and signal transduction in vascular plant primary roots.

During gravitropism, the potential energy of gravity is converted into a biochemical signal. How this transfer occurs remains one of the most exciting mysteries in plant cell biology. New experiments are filling in pieces of the puzzle. In this review, we introduce gravitropism and give an overview of what we know about gravity sensing in roots of vascular plants, with special highlight on recent papers. When plant roots are reoriented sideways, amyloplast resedimentation in the columella cells is a key initial step in gravity sensing. This process somehow leads to cytoplasmic alkalinization of these cells followed by relocalization of auxin efflux carriers (PINs). This changes auxin flow throughout the root, generating a lateral gradient of auxin across the cap that upon transmission to the elongation zone leads to differential cell elongation and gravibending. We will present the evidence for and against the following players having a role in transferring the signal from the amyloplast sedimentation into the auxin signaling cascade: mechanosensitive ion channels, actin, calcium ions, inositol trisphosphate, receptors/ligands, ARG1/ARL2, spermine, and the TOC complex. We also outline auxin transport and signaling during gravitropism.

Molecular evolution, functional variation, and proposed nomenclature of the gene family that includes sphingomyelinase D in sicariid spider venoms.

The venom enzyme sphingomyelinase D (SMase D) in the spider family Sicariidae (brown or fiddleback spiders [Loxosceles] and six-eyed sand spiders [Sicarius]) causes dermonecrosis in mammals. SMase D is in a gene family with multiple venom-expressed members that vary in functional specificity. We analyze molecular evolution of this family and variation in SMase D activity among crude venoms using a data set that represents the phylogenetic breadth of Loxosceles and Sicarius. We isolated a total of 190 nonredundant nucleotide sequences encoding 168 nonredundant amino acid sequences of SMase D homologs from 21 species. Bayesian phylogenies support two major clades that we name alpha and beta, within which we define seven and three subclades, respectively. Sequences in the alpha clade are exclusively from New World Loxosceles and Loxosceles rufescens and include published genes for which expression products have SMase D and dermonecrotic activity. The beta clade includes paralogs from New World Loxosceles that have no, or reduced, SMase D and no dermonecrotic activity and also paralogs from Sicarius and African Loxosceles of unknown activity. Gene duplications are frequent, consistent with a birth-and-death model, and there is evidence of purifying selection with episodic positive directional selection. Despite having venom-expressed SMase D homologs, venoms from New World Sicarius have reduced, or no, detectable SMase D activity, and Loxosceles in the Southern African spinulosa group have low SMase D activity. Sequence conservation mapping shows >98% conservation of proposed catalytic residues of the active site and around a plug motif at the opposite end of the TIM barrel, but alpha and beta clades differ in conservation of key residues surrounding the apparent substrate binding pocket. Based on these combined results, we propose an inclusive nomenclature for the gene family, renaming it SicTox, and discuss emerging patterns of functional diversification.

Robust resting state fMRI processing for studies on typical brain development based on multi-echo EPI acquisition.

Several methodological challenges affect the study of typical brain development based on resting state blood oxygenation level dependent (BOLD) functional MRI (fMRI). One such challenge is mitigating artifacts such as those from head motion, known to be more substantial in younger subjects than older subjects. Other challenges include controlling for potential age-dependence in cerebrospinal fluid (CSF) volume affecting anatomical-functional coregistration; in vascular density affecting BOLD contrast-to-noise; and in CSF pulsation creating time series artifacts. Historically, these confounds have been approached through incorporating artifact-specific temporal and/or spatial filtering into preprocessing pipelines. However, such paths often come with new confounds or limitations. In this study we take the approach of a bottom-up revision of fMRI methodology based on acquisition of multi-echo fMRI and comprehensive utilization of the information in the TE-domain to enhance several aspects of fMRI analysis in the context of a developmental study. We show in a cohort of 25 healthy subjects, aged 9 to 43 years, that the analysis of multi-echo fMRI data eliminates a number of arbitrary processing steps such as bandpass filtering and spatial smoothing, while enabling procedures such as [Formula: see text] mapping, BOLD contrast normalization and signal dropout recovery, precise anatomical-functional coregistration based on [Formula: see text] measurements, automatic denoising through removing subject motion, scanner-related signal drifts and physiology, as well as statistical inference for seed-based connectivity. These enhancements are of both theoretical significance and practical benefit in the study of typical brain development.

CACTIN is an essential nuclear protein in Arabidopsis and may be associated with the eukaryotic spliceosome.

CACTIN is a conserved eukaryotic protein without known functional domains. Previous research revealed that CACTIN is essential in animals and protists and that it may function in inflammation pathways in animals; however, these pathways are not as broadly conserved as CACTIN. Therefore, the ancestral molecular function of CACTIN remains unknown. Our studies using Arabidopsis show that CACTIN is required for embryogenesis. Fluorescently tagged CACTIN localizes to nuclear speckles and colocalizes with known splicing proteins. In yeast-two-hybrid studies, we found that CACTIN binds to a putative component of the spliceosome. These findings support a possible role for CACTIN in splicing.

Low copper and high manganese levels in prion protein plaques.

Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques in situ. In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system.

Multiple roles for membrane-associated protein trafficking and signaling in gravitropism.

Gravitropism is a process that allows plant organs to guide their growth relative to the gravity vector. It requires them to sense changes in their orientation and generate a biochemical signal that they transmit to the tissues that drive organ curvature. Trafficking between the plasma membrane and endosomal compartments is important for all of these phases of the gravitropic response. The sedimentation of starch-filled organelles called amyloplasts plays a key role in sensing reorientation, and vacuolar integrity is required for amyloplast sedimentation in shoots. Other proteins associated with the vesicle trafficking pathway contribute to early gravity signal transduction independently of amyloplast sedimentation in both roots and hypocotyls. Phosphatidylinositol signaling, which starts at the plasma membrane and later affects the localization of auxin efflux facilitators, is a likely second messenger in the signal transduction phase of gravitropism. Finally, membrane-localized auxin influx and efflux facilitators contribute to a differential auxin gradient across the gravistimulated organs, which directs root curvature.

Molecular mechanisms of root gravity sensing and signal transduction.

Plants use gravity as a guide to direct their roots down into the soil to anchor themselves and to find resources needed for growth and development. In higher plants, the columella cells of the root tip form the primary site of gravity sensing, and in these cells the sedimentation of dense, starch-filled plastids (amyloplasts) triggers gravity signal transduction. This generates an auxin gradient across the root cap that is transmitted to the elongation zone where it promotes differential cell elongation, allowing the root to direct itself downward. It is still not well understood how amyloplast sedimentation leads to auxin redistribution. Models have been proposed to explain how mechanosensitive ion channels or ligand-receptor interactions could connect these events. Although their roles are still unclear, possible second messengers in this process include protons, Ca(2+), and inositol 1,4,5-triphosphate. Upon gravistimulation, the auxin efflux facilitators PIN3 and PIN7 relocalize to the lower side of the columella cells and mediate auxin redistribution. However, evidence for an auxin-independent secondary mechanism of gravity sensing and signal transduction suggests that this physiological process is quite complex. Furthermore, plants must integrate a variety of environmental cues, resulting in multifaceted relationships between gravitropism and other directional growth responses such as hydro-, photo-, and thigmotropism.