Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for gene delivery systems into animal and plant cells. A non-viral gene carrier containing a cell-penetrating peptide and a cationic sequence was previously developed for use in intact plants and plant cells; however, the permeation pathway of the gene carrier into plant cells is yet to be elucidated, which would facilitate the improvement of the gene delivery efficiency. Here, we identified the vacuum/compression infiltration-mediated permeation pathway of a non-viral gene carrier into plant tissues and cells using a complex of plasmid DNA and a peptide-based gene carrier. This complex was taken up via the hydathodes in Arabidopsis thaliana, and from root hairs in Nicotiana benthamiana. Remarkably, these structurally weak tissues are also routes of bacterial invasion in nature, suggesting that peptide-pDNA complexes invade intact plants through similar pathways as bacterial pathogens.
The balance between carbon and nitrogen is a key determinant of seed storage components, and thus, is of great importance to rice and other seed-based food crops. To clarify the influence of the rhizosphere carbon/nitrogen balance during the maturation stage of several seed components, transcriptome analysis was performed on the seeds from rice plants that were provided additional nitrogen fertilization at heading time. As a result, it was assessed that genes associated with molecular processes such as photosynthesis, trehalose metabolism, carbon fixation, amino acid metabolism, and cell wall metabolism were differentially expressed. Moreover, cellulose and sucrose synthases, which are involved in cellulose synthesis, were down-regulated. Therefore, we compared cellulose content of mature seeds that were treated with additional nitrogen fertilization with those from control plants using calcofluor staining. In these experiments, cellulose content in endosperm from plants receiving additional nitrogen fertilization was less than that in control endosperm. Other starch synthesis-related genes such as starch synthase 1, starch phosphorylase 2, and branching enzyme 3 were also down-regulated, whereas some α-amylase and β-amylase genes were up-regulated. On the other hand, mRNA expression of amino acid biosynthesis-related molecules was up-regulated. Moreover, additional nitrogen fertilization caused accumulation of storage proteins and up-regulated Cys-poor prolamin mRNA expression. These data suggest that additional nitrogen fertilization at heading time changes the expression of some storage substance-related genes and reduces cellulose levels in endosperm.
Different organelles function coordinately in numerous intracellular processes. Photorespiration incidental to photosynthetic carbon fixation is organized across three subcellular compartments: chloroplasts, peroxisomes, and mitochondria. Under light conditions, these three organelles often form a ternary organellar complex in close proximity, suggesting a connection with metabolism during photorespiration. However, due to the heterogeneity of intercellular organelle localization and morphology, organelles' responses to changes in the external environment remain poorly understood. Here we used array tomography by field emission scanning electron microscopy to image organelles inside the whole plant cell at nanometer resolution, generating a three-dimensional (3D) spatial map of the light-dependent positioning of chloroplasts, peroxisomes, nuclei, and vacuoles. Our results show, in light-treated cells, the volume of peroxisomes increased, and mitochondria were simplified. In addition, the population of free organelles decreased, and the ternary complex centered on chloroplasts increased. Moreover, our results emphasized the expansion of the proximity area rather than the increase in the number of proximity sites inter-organelles. All of these phenomena were quantified for the first time on the basis of nanoscale spatial maps. In summary, we provide the first 3D reconstruction of Arabidopsis mesophyll cells, together with nanoscale quantified organelle morphology and their positioning via proximity areas, and then evidence of their light-dependent changes.
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