ATG5-knockout mutants of Physcomitrella provide a platform for analyzing the involvement of autophagy in senescence processes in plant cells

<p>Autophagy is a pathway in which a cell degrades part of its cytoplasm in vacuoles or lysosomes. To identify the physiological functions of autophagy in plants, we disrupted <i>ATG5</i>, an autophagy-related gene, in <i>Physcomitrella</i>, and confirmed that <i>atg5</i> mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, <i>atg5</i> colonies turned yellow earlier than the wild-type (WT) colonies, showing that <i>Physcomitrella atg5</i> mutants, like yeast and <i>Arabidopsis</i>, are sensitive to nutrient starvation. In the dark, even under nutrient-sufficient conditions, colonies turned yellow and the net degradation of chlorophyll and Rubisco protein occurred together with the upregulation of several senescence-associated genes. Yellowing reactions were inhibited by the protein synthesis inhibitor cycloheximide, suggesting that protonemal colonies undergo dark-induced senescence like the green leaves of higher plants. Such senescence responses in the dark occurred earlier in <i>atg5</i> colonies than WT colonies. The sugar content was almost the same between WT and <i>atg5</i> colonies, indicating that the early-senescence phenotype of <i>atg5</i> is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between <i>atg5</i> and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the <i>atg5</i> mutants, irrespective of the early degradation of Rubisco protein. On nutrient-sufficient medium supplemented with casamino acids, the early-senescence phenotype was slightly moderated. We propose that the early-senescence phenotype in <i>atg5</i> mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in <i>Physcomitrella</i>.</p

Photosensitizer-Conjugated Ultrasmall Carbon Nanodots as Multifunctional Fluorescent Probes for Bioimaging

Highly luminescent ultrasmall carbon nanodots (CDs) have been prepared by one-step microwave-assisted pyrolysis and functionalized with fluorescein photosensitizer by a diazo-bond. The absorption edge of such prepared fluorescein–NN–CDs was red-shifted in comparison with the bare one. Nevertheless, the emission signal induced by the nanoparticle quantum-sized graphite structure was quenched due to photoisomerization of the diazo group at the photoexcited state. In order to restrict the photoisomerization, i.e., rotation around the nitrogen–nitrogen bond, the diazo group was fixed by a metal cation to form a complex compound or chelate. The obtained metal complex of fluorescein–NN–CDs shows an absorbance maximum the same as bare CDs but a recovered emission signal from the nanoparticle moiety, which was bathochromically shifted. They exhibit lower quantum yield in comparison with the bare CDs but better photostability toward emission quenching in nutrition cell culture. The formed photosensitizer-conjugated nanoprobes were proposed as multifunctional fluorophores for intracellular <i>in vivo</i> imaging due to their attractive photophysical attributes and tunable and excitation-dependent emission. The bioapplication of photosensitizer-conjugated CDs was demonstrated as fluorescent tracers for endocytosis pathways in cultured Tobacco cells. Their successful staining and lower toxicity to the plant cells were compared with conventional quantum dots (CdSe/ZnS core–shell type, which caused an acute toxicological <i>in vivo</i> effect)

Photosensitizer-Conjugated Ultrasmall Carbon Nanodots as Multifunctional Fluorescent Probes for Bioimaging

Highly luminescent ultrasmall carbon nanodots (CDs) have been prepared by one-step microwave-assisted pyrolysis and functionalized with fluorescein photosensitizer by a diazo-bond. The absorption edge of such prepared fluorescein–NN–CDs was red-shifted in comparison with the bare one. Nevertheless, the emission signal induced by the nanoparticle quantum-sized graphite structure was quenched due to photoisomerization of the diazo group at the photoexcited state. In order to restrict the photoisomerization, i.e., rotation around the nitrogen–nitrogen bond, the diazo group was fixed by a metal cation to form a complex compound or chelate. The obtained metal complex of fluorescein–NN–CDs shows an absorbance maximum the same as bare CDs but a recovered emission signal from the nanoparticle moiety, which was bathochromically shifted. They exhibit lower quantum yield in comparison with the bare CDs but better photostability toward emission quenching in nutrition cell culture. The formed photosensitizer-conjugated nanoprobes were proposed as multifunctional fluorophores for intracellular <i>in vivo</i> imaging due to their attractive photophysical attributes and tunable and excitation-dependent emission. The bioapplication of photosensitizer-conjugated CDs was demonstrated as fluorescent tracers for endocytosis pathways in cultured Tobacco cells. Their successful staining and lower toxicity to the plant cells were compared with conventional quantum dots (CdSe/ZnS core–shell type, which caused an acute toxicological <i>in vivo</i> effect)

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H⁺-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

<p>Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.</p

Plant Cell Wall-Penetrable, Redox-Responsive Silica Nanoprobe for the Imaging of Starvation-Induced Vesicle Trafficking

Autophagy is a self-protection process against reactive oxygen species (ROS). The intracellular level of ROS increased when cells were cultured under nutrient starvation. Antioxidants such as glutathione and ascorbic acid play an important role in ROS removal. However, the cellular redox state in the autophagic pathway is still unclear. Herein, we developed a new redox-sensitive probe with a disulfide-linked silica scaffold to enable the sensing of the reduction environment in cell organelles. This redox-responsive silica nanoprobe (ReSiN) could penetrate the plant cell wall and release fluorescent molecules in response to redox states. By applying the ReSiN to tobacco BY-2 cells and tracing the distribution of fluorescence, we found a higher reducing potential in the central vacuole than in the autolysosomes. Upon cysteine protease inhibitor (E64-c) treatment in sucrose-free medium, the disulfide-silica structures of the ReSiNs were broken down in the vacuoles but were not degraded and were accumulated in the autolysosomes. These results reveal the feasibility of our nanoprobe for monitoring the endocytic and macroautophagic pathways. These pathways merge upstream of the central vacuole, which is the final destination of both pathways. In addition, different redox potentials were observed in the autophagic pathway. Finally, the expression of the autophagy-related protein (Atg8) fused with green fluorescence protein confirmed that the ReSiN treatment itself did not induce the autophagic pathway under normal physiological conditions, indicating the versatility of this nanoprobe in studying stimuli-triggered autophagy-related trafficking