N6-adenosine methylation (m6A) is a prevalent form of RNA modification found in the expressed transcripts of many eukaryotic organisms. Moreover, m6A methylation is a dynamic and reversible process that requires the functioning of various proteins and their complexes that are evolutionarily conserved between species and include methylases, demethylases, and m6A-binding proteins. Over the past decade, the m6A methylation process in plants has been extensively studied and the understanding thereof has drastically increased, although the regulatory function of some components relies on information derived from animal systems. Notably, m6A has been found to be involved in a variety of factors in RNA processing, such as RNA stability, alternative polyadenylation, and miRNA regulation. The circadian clock in plants is a molecular timekeeping system that regulates the daily and rhythmic activity of many cellular and physiological processes in response to environmental changes such as the day-night cycle. The circadian clock regulates the rhythmic expression of genes through post-transcriptional regulation of mRNA. Recently, m6A methylation has emerged as an additional layer of post-transcriptional regulation that is necessary for the proper functioning of the plant circadian clock. In this review, we have compiled and summarized recent insights into the molecular mechanisms behind m6A modification and its various roles in the regulation of RNA. We discuss the potential role of m6A modification in regulating the plant circadian clock and outline potential future directions for the study of mRNA methylation in plants. A deeper understanding of the mechanism of m6A RNA regulation and its role in plant circadian clocks will contribute to a greater understanding of the plant circadian clock.

Leaf senescence is the final stage of leaf development preceding death, which involves a significant cellular metabolic transition from anabolism to catabolism. Several processes during leaf senescence require coordinated regulation by senescence regulatory genes. In this study, we developed a rapid and systematic cellular approach to dissect the functional roles of genes in senescence regulation through their transient expression in Arabidopsis protoplasts. We established and validated this system by monitoring the differential expression of a luciferase-based reporter that was driven by promoters of SEN4 and SAG12, early and late senescence-responsive genes, depending on effectors of known positive and negative senescence regulators. Overexpression of positive senescence regulators, including ORE1, RPK1, and RAV1, increased the expression of both SEN4- and SAG12-LUC while ORE7, a negative senescence regulator decreased their expression. Consistently with overexpression, knockdown of target genes using amiRNAs resulted in opposite SAG12-LUC expression patterns. The timing and patterns of reporter responses induced by senescence regulators provided molecular evidence for their distinct kinetic involvement in leaf senescence regulation. Remarkably, ORE1 and RPK1 are involved in cell death responses, with more prominent and earlier involvement of ORE1 than RPK1. Consistent with the results in protoplasts, further time series of reactive oxygen species (ROS) and cell death assays using different tobacco transient systems reveal that ORE1 causes acute cell death and RPK1 mediates superoxide-dependent intermediate cell death signaling during leaf senescence. Overall, our results indicated that the luciferase-based reporter system in protoplasts is a reliable experimental system that can be effectively used to examine the regulatory roles of Arabidopsis senescence-associated genes.

The lawn grass Zoysia japonica is widely cultivated for its ornamental and recreational value. However, its green period is subject to shortening, which significantly decreases the economic value of Z. japonica, especially for large cultivations. Leaf senescence is a crucial biological and developmental process that significantly influences the lifespan of plants. Moreover, manipulation of this process can improve the economic value of Z. japonica by extending its greening period. In this study, we conducted a comparative transcriptomic analysis using high-throughput RNA sequencing (RNA-seq) to investigate early senescence responses triggered by age, dark, and salt. Gene set enrichment analysis results indicated that while distinct biological processes were involved in each type of senescence response, common processes were also enriched across all senescence responses. The identification and validation of differentially expressed genes (DEGs) via RNA-seq and quantitative real-time PCR provided up- and down-regulated senescence markers for each senescence and putative senescence regulators that trigger common senescence pathways. Our findings revealed that the NAC, WRKY, bHLH, and ARF transcription factor (TF) groups are major senescence-associated TF families that may be required for the transcriptional regulation of DEGs during leaf senescence. In addition, we experimentally validated the senescence regulatory function of seven TFs including ZjNAP, ZjWRKY75, ZjARF2, ZjNAC1, ZjNAC083, ZjARF1, and ZjPIL5 using a protoplast-based senescence assay. This study provides new insight into the molecular mechanisms underlying Z. japonica leaf senescence and identifies potential genetic resources for enhancing its economic value by prolonging its green period.

Transient expression of genes in protoplasts is a versatile technique for rapid functional characterization of genes by assessing protein localization or effector-reporter responses. In addition, protoplasts have been widely used for generating transgenic or gene-edited plants in model or crop plants. Zoysiagrass (Zoysia japonica Steud.) is one of the most economically important turf plants used in many living places or natural fields, but its management is labor-intensive. Therefore, genetic manipulation using valuable genes is highly demanded in zoysiagrass. Although transient expression systems in zoysiagrass facilitates the identification of valuable zoysiagrass genes, transient expression use in the zoysiagrass protoplasts is yet to be reported. Here we describe the methodology and feasibility for transient expression of genes in the zoysiagrass protoplasts isolated from green leaves. We obtained more than 70% of transfection efficiency in the zoysiagrass protoplasts using polyethylene glycolmediated transfection. Additionally, we showed the feasibility of cellular, biochemical, and molecular approaches using the zoysiagrass protoplasts transiently expressing genes of interest through fluorescent microscopic observation, immunoblot analysis, and luciferase-based promoter activity assay. Along with the genome draft of the Zoysia family, transient expression will be a valuable tool to rapidly investigate gene functions. An initial assessment of gene function using protoplasts facilitate the identification of valuable genes that can improve the economic value of the Z. japonica or its closely related species by gene manipulation.

Polygonum cuspidatum Sieb. et Zucc, a medicinal plant, contains many phenolic compounds such as resveratrol. It has antioxidant, antibacterial, anti-cancer, HIV, and neuron protective properties. In this study, stem segments having an auxiliary bud were cultured in vitro on MS medium supplemented with TDZ at 0.1 mg/L for obtaining the highest number of shoots (8–9 shoots/cluster after eight weeks). The bud shoots were originated from the differentiation in the periphery of the lateral meristem. After eight weeks, the shoots cultured under the monochromatic light showed that the plant height, fresh and dry weight was higher than those grown under the fluorescent light at the same intensity of 50 µmol photon/m2/sec. Blue LED reduced the number of shoots/cluster, and respiration intensity of the inoculum compared to the red LED or fluorescent light conditions. However, after eight weeks, the total sugar and phenolic content in leaves and stems, as well as the resveratrol content of shoots under blue light, were always higher than under red LED or fluorescent light. Moreover, when samples were exposed under the one-week blue light condition at a double intensity (100 µmol photon/m2/sec), the shoot clusters growing under blue LED also had a remarkably high total phenolic content and significantly high resveratrol levels compared to the two other treatments. The roles of endogenous growth regulators in shoot clusters under different lighting conditions were also analyzed and discussed.