Abstract:The sequencing of the moss Physcomitrella patens genome has facilitated studies of the plant proteome. To develop a proteome reference map based on the genome sequence, we conducted 2D electrophoreses of proteins extracted from moss protoplasts, protonemata, and gametophores grown under standard conditions on Petri dishes. On silver-stained gels, depending on the developmental stage of the moss, we resolved from 500 to 600 protein spots that were then excised and digested by trypsin, and 212 proteins were iden… Show more
“…6 ). However, the amount of the large subunit of Rubisco (52.6 kD), which is the most abundant polypeptide in the protonemata of Physcomitrella , 30 decreased rapidly and drastically so that the net degradation for 7 d amounted to more than 70% of the initial amount in atg5 mutants, whereas it decreased to a small extent in WT plants ( Fig. 6 ) Figure 6.…”
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 ATG5, an autophagy-related gene, in Physcomitrella, and confirmed that atg5 mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, atg5 colonies turned yellow earlier than the wild-type (WT) colonies, showing that Physcomitrella atg5 mutants, like yeast and Arabidopsis, 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 atg5 colonies than WT colonies. The sugar content was almost the same between WT and atg5 colonies, indicating that the early-senescence phenotype of atg5 is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between atg5 and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the atg5 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 atg5 mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in Physcomitrella.
“…6 ). However, the amount of the large subunit of Rubisco (52.6 kD), which is the most abundant polypeptide in the protonemata of Physcomitrella , 30 decreased rapidly and drastically so that the net degradation for 7 d amounted to more than 70% of the initial amount in atg5 mutants, whereas it decreased to a small extent in WT plants ( Fig. 6 ) Figure 6.…”
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 ATG5, an autophagy-related gene, in Physcomitrella, and confirmed that atg5 mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, atg5 colonies turned yellow earlier than the wild-type (WT) colonies, showing that Physcomitrella atg5 mutants, like yeast and Arabidopsis, 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 atg5 colonies than WT colonies. The sugar content was almost the same between WT and atg5 colonies, indicating that the early-senescence phenotype of atg5 is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between atg5 and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the atg5 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 atg5 mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in Physcomitrella.
“…Chloroplasts were isolated from protoplasts as previously described [ 27 ]. In short, protoplasts were resuspended in buffer A (50 mM HEPES-KOH, pH 7.5, 330 mM sorbitol, 2 mM EDTA, and 0.4 mM phenylmethylsulfonyl fluoride) and filtered through a double layer of Miracloth (Calbiochem Behring, La Jolla, CA, USA).…”
BackgroundProtein degradation is a basic cell process that operates in general protein turnover or to produce bioactive peptides. However, very little is known about the qualitative and quantitative composition of a plant cell peptidome, the actual result of this degradation. In this study we comprehensively analyzed a plant cell peptidome and systematically analyzed the peptide generation process.ResultsWe thoroughly analyzed native peptide pools of Physcomitrella patens moss in two developmental stages as well as in protoplasts. Peptidomic analysis was supplemented by transcriptional profiling and quantitative analysis of precursor proteins. In total, over 20,000 unique endogenous peptides, ranging in size from 5 to 78 amino acid residues, were identified. We showed that in both the protonema and protoplast states, plastid proteins served as the main source of peptides and that their major fraction formed outside of chloroplasts. However, in general, the composition of peptide pools was very different between these cell types. In gametophores, stress-related proteins, e.g., late embryogenesis abundant proteins, were among the most productive precursors. The Driselase-mediated protonema conversion to protoplasts led to a peptide generation “burst”, with a several-fold increase in the number of components in the latter. Degradation of plastid proteins in protoplasts was accompanied by suppression of photosynthetic activity.ConclusionWe suggest that peptide pools in plant cells are not merely a product of waste protein degradation, but may serve as important functional components for plant metabolism. We assume that the peptide “burst” is a form of biotic stress response that might produce peptides with antimicrobial activity from originally functional proteins. Potential functions of peptides in different developmental stages are discussed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0468-7) contains supplementary material, which is available to authorized users.
“…Making use of this genetic resource, several proteomic datasets were acquired in the last two decades giving insights into abiotic stress and desiccation tolerance‐associated proteins in protonema and gametophores (Cho et al., 2006; Cui et al., 2012; Luo et al., 2020; Mamaeva et al., 2022; Sarnighausen et al., 2004; Skripnikov et al., 2009; Toshima et al., 2014; Wang et al., 2010; Wang, Yang, Zhang, et al., 2009; Wang, Yang, Liu, et al., 2009; Wang et al., 2008; Yotsui et al., 2016).…”
SUMMARYThe establishment of moss spores is considered a milestone in plant evolution. They harbor protein networks underpinning desiccation tolerance and accumulation of storage compounds that can be found already in algae and that are also utilized in seeds and pollen. Furthermore, germinating spores must produce proteins that drive the transition through heterotrophic growth to the autotrophic plant. To get insight into the plasticity of this proteome, we investigated it at five timepoints of moss (Physcomitrium patens) spore germination and in protonemata and gametophores. The comparison to previously published Arabidopsis proteome data of seedling establishment showed that not only the proteomes of spores and seeds are functionally related, but also the proteomes of germinating spores and young seedlings. We observed similarities with regard to desiccation tolerance, lipid droplet proteome composition, control of dormancy, and β‐oxidation and the glyoxylate cycle. However, there were also striking differences. For example, spores lacked any obvious storage proteins. Furthermore, we did not detect homologs to the main triacylglycerol lipase in Arabidopsis seeds, SUGAR DEPENDENT1. Instead, we discovered a triacylglycerol lipase of the oil body lipase family and a lipoxygenase as being the overall most abundant proteins in spores. This finding indicates an alternative pathway for triacylglycerol degradation via oxylipin intermediates in the moss. The comparison of spores to Nicotiana tabacum pollen indicated similarities for example in regards to resistance to desiccation and hypoxia, but the overall developmental pattern did not align as in the case of seedling establishment and spore germination.
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