Tomato strigolactones: A more detailed look

Strigolactones are plant signaling molecules that induce germination of parasitic plant seeds, initiate host plant - arbuscular mycorrhizal fungus symbiosis and act as plant hormones controlling shoot branching and root architecture. To date four unique strigolactones (e.g., orobanchol, didehydroorobanchol isomers 1 and 2 and the aromatic strigolactone solanacol) have been reported in the root exudates and extracts of tomato (Solanum lycopersicum). Here we report on the presence of several additional strigolactones in tomato root exudates and extracts, orobanchyl acetate, two 7-hydroxyorobanchol isomers, 7-oxoorobanchol and two additional didehydroorobanchol isomers and discuss their possible biological relevance.     

Klotho Sensitivity of the Neuronal Excitatory Amino Acid Transporters EAAT3 and EAAT4

Klotho, a transmembrane protein, which can be cleaved off as β-glucuronidase and hormone, is released in both, kidney and choroid plexus and encountered in blood and cerebrospinal fluid. Klotho deficiency leads to early appearance of age-related disorders and premature death. Klotho may modify transport by inhibiting 1,25(OH)₂D₃ formation or by directly affecting channel and carrier proteins. The present study explored whether Klotho influences the activity of the Na⁺-coupled excitatory amino acid transporters EAAT3 and EAAT4, which are expressed in kidney (EAAT3), intestine (EAAT3) and brain (EAAT3 and EAAT4). To this end, cRNA encoding EAAT3 or EAAT4 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho. EAAT expressing Xenopus oocytes were further treated with recombinant human β-Klotho protein with or without β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). Electrogenic excitatory amino acid transport was determined as L-glutamate-induced current (I_glu) in two electrode voltage clamp experiments. EAAT3 and EAAT4 protein abundance in the Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified utilizing chemiluminescence. As a result, coexpression of Klotho cRNA significantly increased I_glu in both, EAAT3 or EAAT4-expressing Xenopus oocytes. Klotho cRNA coexpression significantly increased the maximal current and cell membrane protein abundance of both EAAT3 and EAAT4. The effect of Klotho coexpression on EAAT3 and EAAT4 activity was mimicked by treating EAAT3 or EAAT4-expressing Xenopus oocytes with recombinant human β-Klotho protein. The effects of Klotho coexpression and of treatment with recombinant human β-Klotho protein were both abrogated in the presence of DSAL (10 µM). In conclusion, Klotho is a novel, powerful regulator of the excitatory amino acid transporters EAAT3 and EAAT4.     

Downregulation of KCNQ4 by Janus Kinase 2

Janus kinase-2 (JAK2) participates in the signaling of several hormones, growth factors and cytokines. Further stimulators of JAK2 include osmotic cell shrinkage, and the kinase activates the cell volume regulatory Na⁺/H⁺ exchanger. The kinase may thus participate in cell volume regulation. Cell shrinkage is known to inhibit K⁺ channels. Volume-regulatory K⁺ channels include the voltage-gated K⁺ channel KCNQ4. The present study explored the effect of JAK2 on KCNQ4 channel activity. KCNQ4 was expressed in Xenopus oocytes with or without wild-type JAK2, constitutively active ^V617FJAK2 or inactive ^K882EJAK2; and cell membrane conductance was determined by dual-electrode voltage clamp. Expression of KCNQ4 was followed by the appearance of voltage-gated K⁺ conductance. Coexpression of JAK2 or of ^V617FJAK2, but not of ^K882EJAK2, resulted in a significant decrease in conductance. Treatment of KCNQ4 and JAK2 coexpressing oocytes with the JAK2 inhibitor AG490 (40 μM) was followed by an increase in conductance. Treatment of KCNQ4 expressing oocytes with brefeldin A (5 μM) was followed by a decrease in conductance, which was similar in oocytes expressing KCNQ4 together with JAK2 as in oocytes expressing KCNQ4 alone. Thus, JAK2 apparently does not accelerate channel protein retrieval from the cell membrane. In conclusion, JAK2 downregulates KCNQ4 activity and thus counteracts K⁺ exit, an effect which may contribute to cell volume regulation.     

Physiological Effects of the Synthetic Strigolactone Analog GR24 on Root System Architecture in Arabidopsis: Another Belowground Role for Strigolactones?

In this study, the role of the recently identified class of phytohormones, strigolactones, in shaping root architecture was addressed. Primary root lengths of strigolactone-deficient and -insensitive Arabidopsis (Arabidopsis thaliana) plants were shorter than those of wild-type plants. This was accompanied by a reduction in meristem cell number, which could be rescued by application of the synthetic strigolactone analog GR24 in all genotypes except in the strigolactone-insensitive mutant. Upon GR24 treatment, cells in the transition zone showed a gradual increase in cell length, resulting in a vague transition point and an increase in transition zone size. PIN1/3/7-green fluorescent protein intensities in provascular tissue of the primary root tip were decreased, whereas PIN3-green fluorescent protein intensity in the columella was not affected. During phosphate-sufficient conditions, GR24 application to the roots suppressed lateral root primordial development and lateral root forming potential, leading to a reduction in lateral root density. Moreover, auxin levels in leaf tissue were reduced. When auxin levels were increased by exogenous application of naphthylacetic acid, GR24 application had a stimulatory effect on lateral root development instead. Similarly, under phosphate-limiting conditions, endogenous strigolactones present in wild-type plants stimulated a more rapid outgrowth of lateral root primordia when compared with strigolactone-deficient mutants. These results suggest that strigolactones are able to modulate local auxin levels and that the net result of strigolactone action is dependent on the auxin status of the plant. We postulate that the tightly balanced auxin-strigolactone interaction is the basis for the mechanism of the regulation of the plants' root-to-shoot ratio.     

Side-chain conformational changes upon Protein-Protein Association

Conformational changes upon protein-protein association are the key element of the binding mechanism. The study presents a systematic large-scale analysis of such conformational changes in the side chains. The results indicate that short and long side chains have different propensities for the conformational changes. Long side chains with three or more dihedral angles are often subject to large conformational transition. Shorter residues with one or two dihedral angles typically undergo local conformational changes not leading to a conformational transition. A relationship between the local readjustments and the equilibrium fluctuations of a side chain around its unbound conformation is suggested. Most of the side chains undergo larger changes in the dihedral angle most distant from the backbone. The frequencies of the core-to-surface interface transitions of six nonpolar residues and Tyr are larger than the frequencies of the opposite surface-to-core transitions. The binding increases both polar and nonpolar interface areas. However, the increase of the nonpolar area is larger for all considered classes of protein complexes, suggesting that the protein association perturbs the unbound interfaces to increase the hydrophobic contribution to the binding free energy. To test modeling approaches to side-chain flexibility in protein docking, conformational changes in the X-ray set were compared with those in the docking decoy sets. The results lead to a better understanding of the conformational changes in proteins and suggest directions for efficient conformational sampling in docking protocols.     

Catalase activity prevents exercise-induced up-regulation of vasoprotective proteins in venous tissue

Physical activity induces favourable changes of arterial gene expression and protein activity, although little is known about its effect in venous tissue. Although our understanding of the initiating molecular signals is still incomplete, increased expression of endothelial nitric oxide synthase (eNOS) is considered a key event. This study sought to investigate the effects of two different training protocols on the expression of eNOS and extracellular superoxide dismutase (ecSOD) in venous and lung tissue and to evaluate the underlying molecular mechanisms. C57Bl/6 mice underwent voluntary exercise or forced physical activity. Changes of vascular mRNA and protein levels and activity of eNOS, ecSOD and catalase were determined in aorta, heart, lung and vena cava. Both training protocols similarly increased relative heart weight and resulted in up-regulation of aortic and myocardial eNOS. In striking contrast, eNOS expression in vena cava and lung remained unchanged. Likewise, exercise up-regulated ecSOD in the aorta and in left ventricular tissue but remained unchanged in lung tissue. Catalase expression in lung tissue and vena cava of exercised mice exceeded that in aorta by 6.9- and 10-fold, respectively, suggesting a lack of stimulatory effects of hydrogen peroxide. In accordance, treatment of mice with the catalase inhibitor aminotriazole for 6 weeks resulted in significant up-regulation of eNOS and ecSOD in vena cava. These data suggest that physiological venous catalase activity prevents exercise-induced up-regulation of eNOS and ecSOD. Furthermore, therapeutic inhibition of vascular catalase might improve pulmonary rehabilitation.     

Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

Background

Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels.

Methodology/Principal Findings

The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases ({"type":"entrez-protein","attrs":{"text":"ORF01222","term_id":"1178790230","term_text":"ORF01222"}}ORF01222, {"type":"entrez-protein","attrs":{"text":"ORF03896","term_id":"1178792974","term_text":"ORF03896"}}ORF03896, and {"type":"entrez-protein","attrs":{"text":"ORF01315","term_id":"1178790325","term_text":"ORF01315"}}ORF01315) exhibited the highest levels of up-regulation.

Conclusions/Significance

The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.