Quantifying the effect of soil moisture content on segmenting root system architecture in X-ray computed tomography images

Aims

A commonly accepted challenge when visualising plant roots in X-ray micro Computed Tomography (μCT) images is the similar X-ray attenuation of plant roots and soil phases. Soil moisture content remains a recognised, yet currently uncharacterised source of segmentation error. This work sought to quantify the effect of soil moisture content on the ability to segment roots from soil in μCT images.

Methods

Rice (Oryza sativa) plants grown in contrasting soils (loamy sand and clay loam) were μCT scanned daily for nine days whilst drying from saturation. Root volumes were segmented from μCT images and compared with volumes derived by root washing.

Results

At saturation the overlapping attenuation values of root material, water-filled soil pores and soil organic matter significantly hindered segmentation. However, in dry soil (ca. six days of drying post-saturation) the air-filled pores increased image noise adjacent to roots and impeded accurate visualisation of root material. The root volume was most accurately segmented at field capacity.

Conclusions

Root volumes can be accurately segmented from μCT images of undisturbed soil without compromising the growth requirements of the plant providing soil moisture content is kept at field capacity. We propose all future studies in this area should consider the error associated with scanning at different soil moisture contents.     

Mesenchyme-specific Knockout of ESET Histone Methyltransferase Causes Ectopic Hypertrophy and Terminal Differentiation of Articular Chondrocytes

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.     

Diacylglycerol Kinase δ Modulates Akt Phosphorylation through Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatase 2 (PHLPP2)

Discovering proteins that modulate Akt signaling has become a critical task, given the oncogenic role of Akt in a wide variety of cancers. We have discovered a novel diacylglycerol signaling pathway that promotes dephosphorylation of Akt. This pathway is regulated by diacylglycerol kinase δ (DGKδ). In DGKδ-deficient cells, we found reduced Akt phosphorylation downstream of three receptor tyrosine kinases. Phosphorylation upstream of Akt was not affected. Our data indicate that PKCα, which is excessively active in DGKδ-deficient cells, promotes dephosphorylation of Akt through pleckstrin homology domain leucine-rich repeats protein phosphatase (PHLPP) 2. Depletion of either PKCα or PHLPP2 rescued Akt phosphorylation in DGKδ-deficient cells. In contrast, depletion of PHLPP1, another Akt phosphatase, failed to rescue Akt phosphorylation. Other PHLPP substrates were not affected by DGKδ deficiency, suggesting mechanisms allowing specific modulation of Akt dephosphorylation. We found that β-arrestin 1 acted as a scaffold for PHLPP2 and Akt1, providing a mechanism for specificity. Because of its ability to reduce Akt phosphorylation, we tested whether depletion of DGKδ could attenuate tumorigenic properties of cultured cells and found that DGKδ deficiency reduced cell proliferation and migration and enhanced apoptosis. We have, thus, discovered a novel pathway in which diacylglycerol signaling negatively regulates Akt activity. Our collective data indicate that DGKδ is a pertinent cancer target, and our studies could lay the groundwork for development of novel cancer therapeutics.     

Contrasting calcium localization and speciation in leaves of the Medicago truncatula mutant cod5 analyzed via synchrotron X–ray techniques

Oxalate‐producing plants accumulate calcium oxalate crystals (CaOx_(c)) in the range of 3–80% w/w of their dry weight, reducing calcium (Ca) bioavailability. The calcium oxalate deficient 5 (cod5) mutant of Medicago truncatula has been previously shown to contain similar Ca concentrations to wild‐type (WT) plants, but lower oxalate and CaOx_(c) concentrations. We imaged the Ca distribution in WT and cod5 leaflets via synchrotron X–ray fluorescence mapping (SXRF). We observed a difference in the Ca distribution between cod5 and WT leaflets, manifested as an abundance of Ca in the interveinal areas and a lack of Ca along the secondary veins in cod5, i.e. the opposite of what is observed in WT. X–ray microdiffraction (μXRD) of M. truncatula leaves confirmed that crystalline CaOx_(c) (whewellite; CaC₂O₄·H₂O) was present in the WT only, in cells sheathing the secondary veins. Together with μXRD, microbeam Ca K–edge X–ray absorption near‐edge structure spectroscopy (μXANES) indicated that, among the forms of CaOx, i.e. crystalline or amorphous, only amorphous CaOx was present in cod5. These results demonstrate that deletion of COD5 changes both Ca localization and the form of CaOx within leaflets.     

Diversification in a biodiversity hot spot: landscape correlates of phylogeographic patterns in the African spotted reed frog

The Eastern Afromontane Biodiversity Hotspot is known for microendemism and exceptional population genetic structure. The region's landscape heterogeneity is thought to limit gene flow between fragmented populations and create opportunities for regional adaptation, but the processes involved are poorly understood. Using a combination of phylogeographic analyses and circuit theory, I investigate how characteristics of landscape heterogeneity including regional distributions of slope, rivers and streams, habitat and hydrological basins (drainages) impact genetic distance among populations of the endemic spotted reed frog (Hyperolius substriatus), identifying corridors of connectivity as well as barriers to dispersal. Results show that genetic distance among populations is most strongly correlated to regional and local hydrologic structure and the distribution of suitable habitat corridors, not isolation by distance. Contrary to expectations, phylogeographic structure is not coincident with the two montane systems, but instead corresponds to the split between the region's two major hydrological basins (Zambezi and East Central Coastal). This results in a paraphyletic relationship for the Malawian Highlands populations with respect to the Eastern Arc Mountains and implies that the northern Malawian Highlands are the diversity centre for H. substriatus. Although the Malawian Highlands collectively hold the greatest genetic diversity, individual populations have lower diversity than their Eastern Arc counterparts, with an overall pattern of decreasing population diversity from north to south. Through the study of intraspecific differentiation across a mosaic of ecosystem and geographic heterogeneity, we gain insight into the processes of diversification and a broader understanding of the role of landscape in evolution.     

Inhibition of Dopamine Transporter Activity by G Protein βγ Subunits

Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson’s disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.