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Leaf senescence accompanied by yellowing and Rubisco degradation occurs prematurely in response to various stresses. However, signaling pathways between stress perception and senescence responses are not understood fully, although previous studies suggest the involvement of reactive oxygen species (ROS). While investigating the physiological functions of autophagy in Physcomitrium patens using wild-type (WT) and autophagy-deficient atg5 strains, we found that Physcomitrium colonies senesce prematurely under dark or nitrogen-deficient conditions, with atg5 senescing earlier than WT. In the present study, we measured cellular H2O2, and examined whether H2O2 mediates premature senescence in Physcomitrium colonies. Methyl viologen, an ROS generator, increased cellular H2O2 levels and caused senescence-like symptoms. H2O2 levels were also elevated to the same plateau levels in WT and atg5 under dark or nitrogen-deficient conditions. The ROS scavenger N-acetylcysteine and the ROS source inhibitor carbonyl cyanide m-chlorophenylhydrazone inhibited the increase in H2O2 levels as well as senescence. Upon transfer to a nitrogen-deficient medium, H2O2 levels increased earlier in atg5 than in WT by ~18 h, whereas atg5 yellowed earlier by >2 days. We conclude that the increased H2O2 levels under dark or nitrogen-deficient conditions mediate premature senescence in Physcomitrium but do not explain the different senescence responses of WT and atg5 cells.

In ecology and population biology, logistic equation is widely applied for simulating the population of organisms. By combining the logistic model with the low-density effect called Allee effect, several variations of mathematical expressions have been proposed. The upper half of the work was dedicated to establish a novel equation for highly flexible density effect model with Allee threshold. Allee effect has been rarely observed in microorganisms with asexual reproduction despite of theoretical studies. According to the exploitation ecosystem hypotheses, plants are believed to be insensitive to Allee effect. Taken together, knowledge on the existence of low-density effect in photosynthetic microorganisms is required for redefining the ecological theories emphasizing the photosynthetic organisms as the basis for food chains. Therefore, in the lower half of the present article, we report on the possible Allee effect in photo-autotrophic organisms, namely, green paramecia, and cyanobacteria. Optically monitored growth of green paramecia was shown to be regulated by Allee-like weak low-density effect under photo-autotrophic and photo-heterotrophic conditions. Insensitiveness of wild type cyanobacteria ( Synechocystis sp. Strain PCC6803) to low-density effect was confirmed, as consistent with our empirical knowledge. In contrast, a mutant line of PCC6803 impaired with a photosynthesis-related pxcA gene was shown to be sensitive to typical Allee's low-density effect ( i.e . this line of cells failed to propagate at low cellular density while cells start logarithmic growth at relatively higher inoculating density). This is the first observation that single-gene mutation in an autotrophic organism alters the sensitivity to Allee effect., Competing Interests: No potential conflict of interest was reported by the authors., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Autolysosomes are organelles that sequester and degrade a portion of the cytoplasm during autophagy. Although autophagosomes are short lived compared to other organelles such as mitochondria, plastids, and peroxisomes, many autolysosomes accumulate in tobacco BY-2 cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We here describe our methodology for isolating autolysosomes from BY-2 cells by conventional cell fractionation using a Percoll gradient. The autolysosome fraction separates clearly from fractions containing mitochondria and peroxisomes. It contains acid phosphatase, vacuolar H + -ATPase, and protease activity. Electron micrographs show that the fraction contains partially degraded cytoplasm seen in autolysosomes before isolation although an autolysosome structure is only partially preserved.

Tobacco culture cells carry out a large-scale degradation of intracellular proteins in order to survive under sucrose starvation conditions. We have previously suggested that this bulk degradation of cellular proteins is performed by autophagy, where autolysosomes formed de novo act as the major lytic compartments. The digestion process in autolysosomes can be retarded by addition of the cysteine protease inhibitor E-64c to the culture medium, resulting in the accumulation of autolysosomes. In the present study, we have investigated several properties of autolysosomes in tobacco cells. Electron microscopy showed that the autolysosomes contain osmiophilic particles, some of which resemble partially degraded mitochondria. It also revealed the presence of two kinds of autolysosome precursor structures; one resembled the isolation membrane and the other the autophagosome of mammalian cells. Immunofluorescence microscopy showed that autolysosomes contain acid phosphatase, in accordance with cytochemical enzyme analyses by light and electron microscopy in a previous study. Autolysosomes isolated by cell fractionation on Percoll gradients showed the localization of acid phosphatase, vacuolar H(+)-ATPase and cysteine protease. These results show that starvation-induced autophagy in tobacco cells follows a macroautophagic-type response similar to that described for other eukaryotes. However, our results indicate that, although the plant vacuole is often described as being equivalent to the lysosome of the animal cell, a new low pH lytic compartment-the autolysosome-also contributes to proteolytic degradation when tobacco cells are subjected to sucrose deprivation.

Tobacco (Nicotiana tabacum) culture cells perform autophagy and degrade cellular proteins in response to sucrose starvation. When protein degradation is blocked by the cysteine protease inhibitor E-64c, lysosomes containing particles of cytoplasm (autolysosomes) accumulate in the cells. Therefore, using light microscopy, we can determine whether cells have performed autophagy. In this study, we investigated whether or not 3-methyladenine (3-MA), which is a known inhibitor of autophagy in mammalian cells, blocks autophagy in tobacco culture cells. The accumulation of autolysosomes was blocked by the addition to the culture media of 5 mM 3-MA together with E-64c. We did not detect autolysosomes or structures thought to be involved with autophagy, such as autophagosomes, accumulating in these cells, as observed by electron microscopy. 3-MA blocked cellular protein degradation without any effect on cellular protease activity. In mammalian cells, phosphatidylinositol 3-kinase (PtdIns 3-kinase) is a putative target of 3-MA. The PtdIns 3-kinase inhibitors wortmannin and LY294002 also inhibited the accumulation of autolysosomes in tobacco culture cells. These results suggest that (1) 3-MA inhibits autophagy by blocking the formation of autophagosomes in tobacco culture cells, and (2) PtdIns 3-kinase is essential for autophagy in tobacco cells.

Studies on developmental changes of ganglioside synthesis and compositions were carried out using rat bone marrow cells, spleen and thymus. Ganglioside synthesis was studied by assaying sialyltransferase for GM3 synthesis and GM1b synthesis. In bone marrow cells, peaks of both enzyme activities occurred coincidentally in 2- to 5-week-old rats. In spleen, the highest activities of these enzymes were observed in one-week-old rats. GM1b synthesis in the thymus was almost constant after birth, but GM3 synthesis could not be detected at any age examined. Developmental changes of gangliosides in these tissues were analyzed by thin layer chromatography. Gangliosides corresponding to GM1b and GM3 were recognized in each tissue. The ganglioside content of the bone marrow cells increased in 2- to 5-week-old rats. Ganglioside corresponding to GM1b was isolated from the bone marrow cells, and its structure was confirmed to be the same as that of GM1b by sequential hydrolysis of the ganglioside with glycosidases. GM3 was a predominant ganglioside in newborn rat spleen. Ganglioside content in the spleen increased during 2-5 weeks after birth and became constant after 9 weeks. In the thymus, more than 10 different gangliosides were discriminated, but significant changes of ganglioside pattern with the progress of development could not be observed. The developmental change of the ganglioside composition coincided well with the change of sialyltransferase activities.