Auxin biosynthesis: Spatial regulation and adaptation to stress

The plant hormone auxin is essential for plant growth and development, controlling both organ development and overall plant architecture. Auxin homeostasis is regulated by coordination of biosynthesis, transport, conjugation, sequestration/storage, and catabolism to optimize concentration-dependent growth responses and adaptive responses to temperature, water stress, herbivory and pathogens. At present, the best defined pathway of auxin biosynthesis is the TAA/YUC route, in which the tryptophan aminotransferases TAA and TAR and YUCCA flavin-dependent monooxygenases produce the auxin indole-3-acetic acid from tryptophan. This review highlights recent advances in our knowledge of TAA/YUC-dependent auxin biosynthesis focussing on membrane localisation of auxin biosynthetic enzymes, differential regulation in root and shoot tissue, and auxin biosynthesis during abiotic stress

Characterisation of intracellular membrane structures derived from a massive expansion of ER membrane due to synthetic ER-membrane-resident polyproteins

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring. Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER-proliferation: interaction of the proteins with each other alters the local structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes leading to the formation organised smooth endoplasmic reticulum (OSER). However, these membrane structures formed by ER proliferation are poorly characterised and this hampers their potential development for plant synthetic biology. Here we characterise a range of ER-derived membranous compartments in tobacco and show how the nature of the polyproteins introduced into the ER membrane affect the final compartment morphology. We show that a cytosol-facing oligomerisation domain is an essential component for compartment formation. Using FRAP, we demonstrate that although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol associated with the compartment. Using quantitative image analysis, we also show that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment and for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally under standard conditions

A novel plant actin-microtubule bridging complex regulates cytoskeletal and ER structure at ER-PM contact sites

In plants, the cortical endoplasmic reticulum (ER) network is connected to the plasma membrane (PM) through the ER-PM contact sites (EPCSs), whose structures are maintained by EPCS resident proteins and the cytoskeleton.1, 2, 3, 4, 5, 6, 7 Strong co-alignment between EPCSs and the cytoskeleton is observed in plants,1,8 but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here, we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) analysis to identify two microtubule binding proteins, KLCR1 (kinesin-light-chain-related protein 1) and IQD2 (IQ67-domain 2), that interact with the actin binding protein NET3C and form a component of plant EPCS that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology and EPCS structure. Their loss-of-function mutants, net3a/NET3C RNAi, klcr1, or iqd2, exhibit defects in pavement cell morphology, which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal-associated complex, which is essential for the maintenance and organization of cytoskeletal structure and ER morphology at the EPCS and for normal plant cell morphogenesis

Structural differences in cortical shell properties between upper and lower human fibula as described by pQCT serial scans.A biomechanical interpretation

This study describes the structural features of fibula cortical shell as allowed by serial pQCT scans in 10/10 healthy men and women aged 20–40 years. Indicators of cortical mass (mineral content -BMC-, cross-sectional area -CSA-), mineralization (volumetric BMD, vBMD), design (perimeters, thickness, moments of inertia -MIs-) and strength (Bone Strength Indices, BSIs; polar Strength-Strain Index, pSSI) were determined. All cross-sectional shapes and geometrical or strength indicators suggested a sequence of five different regions along the bone, which would be successively adapted to 1. transmit loads from the articular surface to the cortical shell (near the proximal tibia-fibular joint), 2. favor lateral bending (central part of upper half), 3. resist lateral bending (mid-diaphysis), 4. favor lateral bending again (central part of the lower half), and 5. resist bending/torsion (distal end). Cortical BMC and the cortical/total CSA ratio were higher at the midshaft than at both bone ends (p < 0.001). However, all MIs, BSIs and pSSI values and the endocortical perimeter/cortical CSA ratio (indicator of the mechanostat's ability to re-distribute the available cortical mass) showed a “W-shaped” distribution along the bone, with maximums at the mid-shaft and at both bone's ends (site effect, p < 0.001). The correlation coefficient (r) of the relationship between MIs (y) and cortical vBMD (x) at each bone site (“distribution/quality” curve that describes the efficiency of distribution of the cortical tissue as a function of the local tissue stiffness) was higher at proximal than distal bone regions (p < 0.001). The results from the study suggest that human fibula is primarily adapted to resist bending and torsion rather than compression stresses, and that fibula's bending strength is lower at the center of its proximal and distal halves and higher at the mid-shaft and at both bone's ends. This would favor, proximally, the elastic absorption of energy by the attached muscles that rotate or evert the foot, and distally, the widening of the heel joint and the resistance to excessive lateral bending. Results also suggest that biomechanical control of structural stiffness differs between proximal and distal fibula

A C-terminal amphipathic helix is necessary for the in vivo tubule-shaping function of a plant reticulon

​Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function

The Role of Oligomerization and Cooperative Regulation in Protein Function: The Case of Tryptophan Synthase

The oligomerization/co-localization of protein complexes and their cooperative regulation in protein function is a key feature in many biological systems. The synergistic regulation in different subunits often enhances the functional properties of the multi-enzyme complex. The present study used molecular dynamics and Brownian dynamics simulations to study the effects of allostery, oligomerization and intermediate channeling on enhancing the protein function of tryptophan synthase (TRPS). TRPS uses a set of α/β–dimeric units to catalyze the last two steps of L-tryptophan biosynthesis, and the rate is remarkably slower in the isolated monomers. Our work shows that without their binding partner, the isolated monomers are stable and more rigid. The substrates can form fairly stable interactions with the protein in both forms when the protein reaches the final ligand–bound conformations. Our simulations also revealed that the α/β–dimeric unit stabilizes the substrate–protein conformation in the ligand binding process, which lowers the conformation transition barrier and helps the protein conformations shift from an open/inactive form to a closed/active form. Brownian dynamics simulations with a coarse-grained model illustrate how protein conformations affect substrate channeling. The results highlight the complex roles of protein oligomerization and the fine balance between rigidity and dynamics in protein function

A Novel Intracellular Isoform of Matrix Metalloproteinase-2 Induced by Oxidative Stress Activates Innate Immunity

Experimental and clinical evidence has pinpointed a critical role for matrix metalloproteinase-2 (MMP-2) in ischemic ventricular remodeling and systolic heart failure. Prior studies have demonstrated that transgenic expression of the full-length, 68 kDa, secreted form of MMP-2 induces severe systolic failure. These mice also had unexpected and severe mitochondrial structural abnormalities and dysfunction. We hypothesized that an additional intracellular isoform of MMP-2, which affects mitochondrial function is induced under conditions of systolic failure-associated oxidative stress.Western blots of cardiac mitochondria from the full length MMP-2 transgenics, ageing mice and a model of accelerated atherogenesis revealed a smaller 65 kDa MMP-2 isoform. Cultured cardiomyoblasts subjected to transient oxidative stress generated the 65 kDa MMP-2 isoform. The 65 kDa MMP-2 isoform was also induced by hypoxic culture of cardiomyoblasts. Genomic database analysis of the MMP-2 gene mapped transcriptional start sites and RNA transcripts induced by hypoxia or epigenetic modifiers within the first intron of the MMP-2 gene. Translation of these transcripts yields a 65 kDa N-terminal truncated isoform beginning at M(77), thereby deleting the signal sequence and inhibitory prodomain. Cellular trafficking studies demonstrated that the 65 kDa MMP-2 isoform is not secreted and is present in cytosolic and mitochondrial fractions, while the full length 68 kDa isoform was found only in the extracellular space. Expression of the 65 kDa MMP-2 isoform induced mitochondrial-nuclear stress signaling with activation of the pro-inflammatory NF-κB, NFAT and IRF transcriptional pathways. By microarray, the 65 kDa MMP-2 induces an innate immunity transcriptome, including viral stress response genes, innate immunity transcription factor IRF7, chemokines and pro-apoptosis genes.A novel N-terminal truncated intracellular isoform of MMP-2 is induced by oxidative stress. This isoform initiates a primary innate immune response that may contribute to progressive cardiac dysfunction in the setting of ischemia and systolic failure

An improved wavelet-ARIMA approach for forecasting metal prices

Metal price forecasts support estimates of future profits from metal exploration and mining and inform purchasing, selling and other day-to-day activities in the metals industry. Past research has shown that cyclical behaviour is a dominant characteristic of metal prices. Wavelet analysis enables to capture this cyclicality by decomposing a time series into its frequency and time domain. This study assesses the usefulness of an improved combined wavelet-autoregressive integrated moving average (ARIMA) approach for forecasting monthly prices of aluminium, copper, lead and zinc. The performance of ARIMA models in forecasting metal prices is demonstrated to be increased substantially through a wavelet- based multiresolution analysis (MRA) prior to ARIMA model fitting. The approach demonstrated in this paper is novel because it identifies the optimal combination of the wavelet transform type, wavelet function and the number of decomposition levels used in the MRA and thereby increases the forecast accuracy significantly. The results showed that, on average, the proposed framework has the potential to increase the accuracy of one month ahead forecasts by $53/t for aluminium,$126/t for copper, $50/t for lead and$51/t for zinc, relative to classic ARIMA models. This highlights the importance of taking into account cyclicality when forecasting metal prices