Alkali-salinity exerts severe osmotic, ionic, and high-pH stresses to plants. To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species (ROS) homeostasis, physiological and diverse quantitative proteomics analyses of alkaligrass (Puccinellia tenuiflora) under Na2CO3 stress were conducted. In addition, Western blot, real-time PCR, and transgenic techniques were applied to validate the proteomic results and test the functions of the Na2CO3-responsive proteins. A total of 104 and 102 Na2CO3-responsive proteins were identified in leaves and chloroplasts, respectively. In addition, 84 Na2CO3-responsive phosphoproteins were identified, including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts, which are crucial for the regulation of photosynthesis, ion transport, signal transduction, and energy homeostasis. A full-length PtFBA encoding an alkaligrass chloroplastic fructose-bisphosphate aldolase (FBA) was overexpressed in wild-type cells of cyanobacterium Synechocystis sp. Strain PCC 6803, leading to enhanced Na2CO3 tolerance. All these results indicate that thermal dissipation, state transition, cyclic electron transport, photorespiration, repair of photosystem (PS) II, PSI activity, and ROS homeostasis were altered in response to Na2CO3 stress, which help to improve our understanding of the Na2CO3-responsive mechanisms in halophytes.

Background: Salinity has obvious effects on plant growth and crop productivity. The salinity-responsive mechanisms have been well-studied in differentiated organs (e.g., leaves, roots and stems), but not in unorganized cells such as callus. High-throughput quantitative proteomics approaches have been used to investigate callus development, somatic embryogenesis, organogenesis, and stress response in numbers of plant species. However, they have not been applied to callus from monocotyledonous halophyte alkaligrass (Puccinellia tenuifora). Results: The alkaligrass callus growth, viability and membrane integrity were perturbed by 50 mM and 150 mM NaCl treatments. Callus cells accumulated the proline, soluble sugar and glycine betaine for the maintenance of osmotic homeostasis. Importantly, the activities of ROS scavenging enzymes (e.g., SOD, APX, POD, GPX, MDHAR and GR) and antioxidants (e.g., ASA, DHA and GSH) were induced by salinity. The abundance patterns of 55 salt-responsive proteins indicate that salt signal transduction, cytoskeleton, ROS scavenging, energy supply, gene expression, protein synthesis and processing, as well as other basic metabolic processes were altered in callus to cope with the stress. Conclusions: The undifferentiated callus exhibited unique salinity-responsive mechanisms for ROS scavenging and energy supply. Activation of the POD pathway and AsA-GSH cycle was universal in callus and differentiated organs, but salinity-induced SOD pathway and salinity-reduced CAT pathway in callus were different from those in leaves and roots. To cope with salinity, callus mainly relied on glycolysis, but not the TCA cycle, for energy supply.

High temperatures seriously limit plant growth and productivity. Investigating heat-responsive molecular mechanisms is important for breeding heat-tolerant crops. In this study, heat-responsive mechanisms in leaves from a heat-sensitive spinach (Spinacia oleracea L.) variety Sp73 were investigated using two-dimensional gel electrophoresis (2DE)-based and isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics approaches. In total, 257 heat-responsive proteins were identified in the spinach leaves. The abundance patterns of these proteins indicated that the photosynthesis process was inhibited, reactive oxygen species (ROS) scavenging pathways were initiated, and protein synthesis and turnover, carbohydrate and amino acid metabolism were promoted in the spinach Sp73 in response to high temperature. By comparing this with our previous results in the heat-tolerant spinach variety Sp75, we found that heat inhibited photosynthesis, as well as heat-enhanced ROS scavenging, stress defense pathways, carbohydrate and energy metabolism, and protein folding and turnover constituting a conservative strategy for spinach in response to heat stress. However, the heat-decreased biosynthesis of chlorophyll and carotenoid as well as soluble sugar content in the variety Sp73 was quite different from that in the variety Sp75, leading to a lower capability for photosynthetic adaptation and osmotic homeostasis in Sp73 under heat stress. Moreover, the heat-reduced activities of SOD and other heat-activated antioxidant enzymes in the heat-sensitive variety Sp73 were also different from the heat-tolerant variety Sp75, implying that the ROS scavenging strategy is critical for heat tolerance.

The germin-like protein (GLP) family plays vital roles for plant growth, stress adaptation, and defense; however, its evolutionary dynamics and functional diversity in halophytes remain poorly characterized. Here, we present the genome-wide analysis of the GLP family in the halophytic forage alkaligrass (Puccinellia tenuiflora), which identified 54 PutGLPs with a significant expansion compared to other plant species. Phylogenetic analysis revealed monocot-specific clustering, with 41.5% of PutGLPs densely localized to chromosome 7, suggesting tandem duplication as a key driver of family expansion. Collinearity analysis confirmed evolutionary conservation with monocot GLPs. Integrated gene structure and motif analysis revealed conserved cupin domains (BoxB and BoxC). Promoter cis-acting elements analysis revealed stress-responsive architectures dominated by ABRE, STRE, and G-box motifs. Tissue-/organ-specific expression profiling identified root- and flower-enriched PutGLPs, implying specialized roles in stress adaptation. Dynamic expression patterns under salt-dominated stresses revealed distinct regulatory pathways governing ionic and alkaline stress responses. Functional characterization of PutGLP37 demonstrated its cell wall localization, dual superoxide dismutase (SOD) and oxalate oxidase (OXO) enzymatic activities, and salt stress tolerance in Escherichia coli, yeast (Saccharomyces cerevisiae INVSc1), and transgenic Arabidopsis. This study provides critical insights into the evolutionary innovation and stress adaptive roles of GLPs in halophytes.

Background: Nucleosome plays an important role in the process of genome expression, DNA replication, DNA repair and transcription. Therefore, the research of nucleosome positioning has invariably received extensive attention. Considering the diversity of DNA sequence representation methods, we tried to integrate multiple features to analyze its effect in the process of nucleosome positioning analysis. This process can also deepen our understanding of the theoretical analysis of nucleosome positioning. Results: Here, we not only used frequency chaos game representation (FCGR) to construct DNA sequence features, but also integrated it with other features and adopted the principal component analysis (PCA) algorithm. Simultaneously, support vector machine (SVM), extreme learning machine (ELM), extreme gradient boosting (XGBoost), multilayer perceptron (MLP) and convolutional neural networks (CNN) are used as predictors for nucleosome positioning prediction analysis, respectively. The integrated feature vector prediction quality is significantly superior to a single feature. After using principal component analysis (PCA) to reduce the feature dimension, the prediction quality of H. sapiens dataset has been significantly improved. Conclusions: Comparative analysis and prediction on H. sapiens, C. elegans, D. melanogaster and S. cerevisiae datasets, demonstrate that the application of FCGR to nucleosome positioning is feasible, and we also found that integrative feature representation would be better.