Influence of the Stability of a Fused Protein and Its Distance to the Amyloidogenic Segment on Fibril Formation

Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔGD = 12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔGD = 26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation

Plant growth-promoting actinobacteria: a new strategy for enhancing sustainable production and protection of grain legumes

Grain legumes are a cost-effective alternative for the animal protein in improving the diets of the poor in South-East Asia and Africa. Legumes, through symbiotic nitrogen fixation, meet a major part of their own N demand and partially benefit the following crops of the system by enriching soil. In realization of this sustainability advantage and to promote pulse production, United Nations had declared 2016 as the “International Year of pulses”. Grain legumes are frequently subjected to both abiotic and biotic stresses resulting in severe yield losses. Global yields of legumes have been stagnant for the past five decades in spite of adopting various conventional and molecular breeding approaches. Furthermore, the increasing costs and negative effects of pesticides and fertilizers for crop production necessitate the use of biological options of crop production and protection. The use of plant growth-promoting (PGP) bacteria for improving soil and plant health has become one of the attractive strategies for developing sustainable agricultural systems due to their eco-friendliness, low production cost and minimizing consumption of non-renewable resources. This review emphasizes on how the PGP actinobacteria and their metabolites can be used effectively in enhancing the yield and controlling the pests and pathogens of grain legumes

Working day and night

Casein kinase 2 is a ubiquitous protein kinase that has puzzled researchers for several decades because of its pleiotropic activity. Here, we set out to identify the $\textit {in vivo}$ targets of plastid casein kinase 2 (pCK2) in $\textit {Arabidopsis thaliana}$. Survey phosphoproteome analyses were combined with targeted analyses with wild-type and $\textit {pck2}$ knockdown mutants to identify potential pCK2 targets by their decreased phosphorylation state in the mutant. To validate potential substrates, we complemented the $\textit {pck2}$ knockdown line with tandem affinity tag (TAP)-tagged pCK2 and found it to restore growth parameters, as well as many, but not all, putative pCK2-dependent phosphorylation events. We further performed a targeted analysis at the end-of-night to increase the specificity of target protein identification. This analysis confirmed light-independent phosphorylation of several pCK2 target proteins. Based on the aforementioned data, we define a set of $\textit {in vivo}$ pCK2-targets that span different chloroplast functions, such as metabolism, transcription, translation and photosynthesis. The pleiotropy of pCK2 functions is also manifested by altered state transition kinetics during short-term acclimation and significant alterations in the mutant metabolism, supporting its function in photosynthetic regulation. Thus, our data expand our understanding on chloroplast phosphorylation networks and provide insights into kinase networks in the regulation of chloroplast functions

Allene oxide synthase, allene oxide cyclase and jasmonic acid levels in Lotus japonicus nodules.

Jasmonic acid (JA), its derivatives and its precursor cis-12-oxo phytodienoic acid (OPDA) form a group of phytohormones, the jasmonates, representing signal molecules involved in plant stress responses, in the defense against pathogens as well as in development. Elevated levels of JA have been shown to play a role in arbuscular mycorrhiza and in the induction of nitrogen-fixing root nodules. In this study, the gene families of two committed enzymes of the JA biosynthetic pathway, allene oxide synthase (AOS) and allene oxide cyclase (AOC), were characterized in the determinate nodule-forming model legume Lotus japonicus JA levels were to be analysed in the course of nodulation. Since in all L. japonicus organs examined, JA levels increased upon mechanical disturbance and wounding, an aeroponic culture system was established to allow for a quick harvest, followed by the analysis of JA levels in whole root and shoot systems. Nodulated plants were compared with non-nodulated plants grown on nitrate or ammonium as N source, respectively, over a five week-period. JA levels turned out to be more or less stable independently of the growth conditions. However, L. japonicus nodules formed on aeroponically grown plants often showed patches of cells with reduced bacteroid density, presumably a stress symptom. Immunolocalization using a heterologous antibody showed that the vascular systems of these nodules also seemed to contain less AOC protein than those of nodules of plants grown in perlite/vermiculite. Hence, aeroponically grown L. japonicus plants are likely to be habituated to stress which could have affected JA levels.peerReviewe