Integration of Ultrasound into the Development of Plant-Based Protein Hydrolysate and Its Bio-Stimulatory Effect for Growth of Wheat Grain Seedlings In Vivo.

This study was dedicated to increasing the efficiency of producing plant-based protein hydrolysate using traditional and non-traditional treatments. Low- and high frequency ultrasound (US) at different intensities were applied to corn steep liquor (CSL) at 50 °C for 30 min, and enzymatic hydrolysis was performed using industrially produced alkaline protease. The efficiency of US and enzymatic treatments was characterized by protein solubility (soluble protein (SP) content, hydrolyzed protein (HP) concentration, and free amino acid (FAA) profile) and kinetic parameters: Michaelis-Menten constant (KM) and apparent breakdown rate constant (kA). A significant effect of 37 kHz US pre-treatment for CSL enzymatic hydrolysis was found and resulted in the highest HP concentration (17.5 g/L) using the lowest enzyme concentration (2.1 g/L) and the shortest hydrolysis time (60 min). By using US pre-treatment, on average, a 2.2 times higher FAA content could be achieved compared to traditional hydrolysis. Additionally, results for the kinetic parameters kM and kA confirmed the potential of applying US treatment before hydrolysis. The effect of CSL protein hydrolysate on plant growth was tested in vivo on wheat grain seed germination and resulted in the significant increase in germination parameters compared to the control treatment. These findings indicate that by-products of starch industry could be a promising source for the production of low-cost sustainable biostimulants.

Nuclear proteome of virus-infected and healthy potato leaves.

BACKGROUND: Infection of plants by viruses interferes with expression and subcellular localization of plant proteins. Potyviruses comprise the largest and most economically damaging group of plant-infecting RNA viruses. In virus-infected cells, at least two potyviral proteins localize to nucleus but reasons remain partly unknown. RESULTS: In this study, we examined changes in the nuclear proteome of leaf cells from a diploid potato line (Solanum tuberosum L.) after infection with potato virus A (PVA; genus Potyvirus; Potyviridae) and compared the data with that acquired for healthy leaves. Gel-free liquid chromatography-coupled to tandem mass spectrometry was used to identify 807 nuclear proteins in the potato line v2-108; of these proteins, 370 were detected in at least two samples of healthy leaves. A total of 313 proteins were common in at least two samples of healthy and PVA-infected leaves; of these proteins, 8 showed differential accumulation. Sixteen proteins were detected exclusively in the samples from PVA-infected leaves, whereas other 16 proteins were unique to healthy leaves. The protein Dnajc14 was only detected in healthy leaves, whereas different ribosomal proteins, ribosome-biogenesis proteins, and RNA splicing-related proteins were over-represented in the nuclei of PVA-infected leaves. Two virus-encoded proteins were identified in the samples of PVA-infected leaves. CONCLUSIONS: Our results show that PVA infection alters especially ribosomes and splicing-related proteins in the nucleus of potato leaves. The data increase our understanding of potyvirus infection and the role of nucleus in infection. To our knowledge, this is the first study of the nuclear proteome of potato leaves and one of the few studies of changes occurring in nuclear proteomes in response to plant virus infection.

Differential Requirement of the Ribosomal Protein S6 and Ribosomal Protein S6 Kinase for Plant-Virus Accumulation and Interaction of S6 Kinase with Potyviral VPg.

Ribosomal protein S6 (RPS6) is an indispensable plant protein regulated, in part, by ribosomal protein S6 kinase (S6K) which, in turn, is a key regulator of plant responses to stresses and developmental cues. Increased expression of RPS6 was detected in Nicotiana benthamiana during infection by diverse plant viruses. Silencing of the RPS6 and S6K genes in N. benthamiana affected accumulation of Cucumber mosaic virus, Turnip mosaic virus (TuMV), and Potato virus A (PVA) in contrast to Turnip crinkle virus and Tobacco mosaic virus. In addition, the viral genome-linked protein (VPg) of TuMV and PVA interacted with S6K in plant cells, as detected by bimolecular fluorescence complementation assay. The VPg-S6K interaction was detected in cytoplasm, nucleus, and nucleolus, whereas the green fluorescent protein-tagged S6K alone showed cytoplasmic localization only. These results demonstrate that the requirement for RPS6 and S6K differs for diverse plant viruses with different translation initiation strategies and suggest that potyviral VPg-S6K interaction may affect S6K functions in both the cytoplasm and the nucleus.

Application of multiplexed cysteine-labeled complex protein sample for 2D electrophoretic gel alignment.

The analysis of cellular subproteomes by 2DE is hampered by the difficulty of aligning gel images from samples that have very different protein composition. Here, we present a sensitive and cost-effective fluorescent labeling method for analyzing protein samples that is not dependent on their composition. The alignment is guided by inclusion of a complex mixture of proteins that is co-run with the sample. Maleimide-conjugated fluorescent dyes Dy-560 and Dy-635 are used to label the cysteine residues of the sample of interest and the alignment standard, respectively. The two differently labeled mixtures are then combined and separated on a 2D gel and, after selective fluorescence detection, an unsupervised image registration process is used to align the protein patters. In a pilot study, this protocol significantly improved the accuracy of alignment of nuclear proteins with total cellular proteins.