DNA methylation catalyzed by methylase plays an important role in many biological events. However, traditional methods of methylase activity analysis by gel electrophoresis were laborious and discontinuous. In this paper, we report a new strategy to study methylase activity using fluorescent probes coupled with enzyme-linkage reactions. A hairpin DNA probe is prepared with a fluorophore and a quencher linked at the 5'- and 3'-terminus of the probe. A disturbance of the stem sequence by DNA methylation would cause the separation of the fluorophore and the quencher, resulting in the restoration of the fluorescence. We used DNA adenine methylation (Dam) methyltransferase (MTase) and Dpn I endonuclease, both having a 5'-G-A-T-C-3' recognition sequence. Dam MTase catalyzed the methylation of the sequence of 5'-GATC-3', and Dpn I cut the sequence of 5'-G-Am-T-C-3'. The fluorescence of the hairpin probe was restored when it was cleaved by Dpn I endonuclease during the course of methylation. Unlike traditional methods, this assay was done in real time and could be used to monitor the dynamic process of methylation. Our method is easy, simple, and nonradioactive, yet as efficient as gel electrophoresis in detecting the activity of methylase. It also had the potential to screen suitable inhibitor drugs for Dam methylase.

Mitochondria are the source of reactive oxygen species (ROS) in plant cells and play a central role in the mitochondrial electron transport chain (ETC) and tricarboxylic acid cycle (TCA) cycles; however, ROS production and regulation for seed germination, seedling growth, as well as mitochondrial responses to abiotic stress, are not clear. This study was conducted to obtain basic information on seed germination, embryo mitochondrial antioxidant responses, and protein profile changes in artificial aging in oat seeds (Avena sativa L.) exposed to exogenous nitric oxide (NO) treatment. The results showed that the accumulation of H2O2 in mitochondria increased significantly in aged seeds. Artificial aging can lead to a loss of seed vigor, which was shown by a decline in seed germination and the extension of mean germination time (MGT). Seedling growth was also inhibited. Some enzymes, including catalase (CAT), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR), maintained a lower level in the ascorbate-glutathione (AsA-GSH) scavenging system. Proteomic analysis revealed that the expression of some proteins related to the TCA cycle were down-regulated and several enzymes related to mitochondrial ETC were up-regulated. With the application of 0.05 mM NO in aged oat seeds, a protective effect was observed, demonstrated by an improvement in seed vigor and increased H2O2 scavenging ability in mitochondria. There were also higher activities of CAT, GR, MDHAR, and DHAR in the AsA-GSH scavenging system, enhanced TCA cycle-related enzymes (malate dehydrogenase, succinate-CoA ligase, fumarate hydratase), and activated alternative pathways, as the cytochrome pathway was inhibited. Therefore, our results indicated that seedling growth and seed germinability could retain a certain level in aged oat seeds, predominantly depending on the lower NO regulation of the TCA cycle and AsA-GSH. Thus, it could be concluded that the application of 0.05 mM NO in aged oat seeds improved seed vigor by enhancing the mitochondrial TCA cycle and activating alternative pathways for improvement.

The sucrose non-fermentation-related protein kinase (SnRK) is a kind of Ser/Thr protein kinase, which plays a crucial role in plant stress response by phosphorylating the target protein to regulate the interconnection of various signaling pathways. However, little is known about the SnRK family in Eucalyptus grandis. Thirty-four putative SnRK sequences were identified in E. grandis and divided into three subgroups (SnRK1, SnRK2 and SnRK3) based on phylogenetic analysis and the type of domain. Chromosome localization showed that SnRK family members are unevenly distributed in the remaining 10 chromosomes, with the notable exception of chromosome 11. Gene structure analysis reveal that 10 of the 24 SnRK3 genes contained no introns. Moreover, conserved motif analyses showed that SnRK sequences belonged to the same subgroup that contained the same motif type of motif. The Ka/Ks ratio of 17 paralogues suggested that the EgrSnRK gene family underwent a purifying selection. The upstream region of EgrSnRK genes enriched with different type and numbers of cis-elements indicated that EgrSnRK genes are likely to play a role in the response to diverse stresses. Quantitative real-time PCR showed that the majority of the SnRK genes were induced by salt treatment. Genome-wide analyses and expression pattern analyses provided further understanding on the function of the SnRK family in the stress response to different environmental salt concentrations.

Although melatonin has been reported to play an important role in regulating metabolic events under adverse stresses, its underlying mechanisms on germination in aged seeds remain unclear. This study was conducted to investigate the effect of melatonin priming (MP) on embryos of aged oat seeds in relation to germination, ultrastructural changes, antioxidant responses, and protein profiles. Proteomic analysis revealed, in total, 402 differentially expressed proteins (DEPs) in normal, aged, and aged + MP embryos. The downregulated DEPs in aged embryos were enriched in sucrose metabolism, glycolysis, β-oxidation of lipid, and protein synthesis. MP (200 μM) turned four downregulated DEPs into upregulated DEPs, among which, especially 3-ketoacyl-CoA thiolase-like protein (KATLP) involved in the β-oxidation pathway played a key role in maintaining TCA cycle stability and providing more energy for protein translation. Furthermore, it was found that MP enhanced antioxidant capacity in the ascorbate-glutathione (AsA-GSH) system, declined reactive oxygen species (ROS), and improved cell ultrastructure. These results indicated that the impaired germination and seedling growth of aged seeds could be rescued to a certain level by melatonin, predominantly depending on β-oxidation, protein translation, and antioxidant protection of AsA-GSH. This work reveals new insights into melatonin-mediated mechanisms from protein profiles that occur in embryos of oat seeds processed by both aging and priming.