Middle‐Down Characterization of the Cell Cycle Dependence of Histone H4 Posttranslational Modifications and Proteoforms

Post‐translational modifications (PTMs) of histones are important epigenetic regulatory mechanisms that are often dysregulated in cancer. We employ middle‐down proteomics to investigate the PTMs and proteoforms of histone H4 during cell cycle progression. We use pH gradient weak cation exchange‐hydrophilic interaction liquid chromatography (WCX‐HILIC) for on‐line liquid chromatography‐mass spectrometry analysis to separate and analyze the proteoforms of histone H4. This procedure provides enhanced separation of proteoforms, including positional isomers, and simplifies downstream data analysis. We use ultrahigh mass accuracy and resolution Fourier transform‐ion cyclotron resonance (FT‐ICR) mass spectrometer to unambiguously distinguish between acetylation and tri‐methylation (?m = 0.036 Da). In total, we identify and quantify 233 proteoforms of histone H4 in two breast cancer cell lines. We observe significant increases in S1 phosphorylation during mitosis, implicating an important role in mitotic chromatin condensation. A decrease of K20 unmodified proteoforms is observed as the cell cycle progresses, corresponding to an increase of K20 mono‐ and di‐methylation. Acetylation at K5, K8, K12, and K16 declines as cells traverse from S phase to mitosis, suggesting cell cycle–dependence and an important role during chromatin replication and condensation. These new insights into the epigenetics of the cell cycle may provide new diagnostic and prognostic biomarkers.     

Quantitative proteomic analyses reveal the dynamics of protein and amino acid accumulation during soybean seed development

Using high throughput tandem mass tag (TMT) based tagging technique, we identified 4172 proteins in three developmental stages: early, mid, and late seed filling. We mapped the identified proteins to metabolic pathways associated with seed filling. The elevated abundance of several kinases was observed from the early to mid-stages of seed filling, indicating that protein phosphorylation was a significant event during this period. The early to late seed filling stages were characterized by an increased abundance of proteins associated with the cell wall, oil, and vacuolar-related processes. Among the seed storage proteins, 7S (β-subunit) and 11S (Gy3, Gy4, Gy5) steadily increased in abundance during early to late stages of seed filling, whereas 2S albumin exhibited a decrease in abundance during the same period. An increased abundance of proteases, senescence-associated proteins, and oil synthesis proteins was observed from the mid to late seed filling stages. The mid to late stages of seed filling was also characterized by a lower abundance of transferases, transporters, Kunitz family trypsin, and protease inhibitors. Two enzymes associated with methionine synthesis exhibited lower abundance from early to late stages. This study unveiled several essential enzymes/proteins related to amino acid and protein synthesis and their accumulation during seed development. All data can be accessed through this link: https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=38784ecbd0854bb3801afc0d89056f84 . (Accession MSV000087577)     

Evidence for phosphorylation of the major seed storage protein of the common bean and its phosphorylation-dependent degradation during germination

Phaseolin is the major seed storage protein of common bean, Phaseolus vulgaris L., accounting for up to 50 % of the total seed proteome. The regulatory mechanisms responsible for the synthesis, accumulation and degradation of phaseolin in the common bean seed are not yet sufficiently known. Here, we report on a systematic study in dormant and 4-day germinating bean seeds from cultivars Sanilac (S) and Tendergreen (T) to explore the presence and dynamics of phosphorylated phaseolin isoforms. High-resolution two-dimensional electrophoresis in combination with the phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescent stain and chemical dephosphorylation by hydrogen fluoride–pyridine enabled us to identify differentially phosphorylated phaseolin polypeptides in dormant and germinating seeds from cultivars S and T. Phosphorylated forms of the two subunits of type α and β that compose the phaseolin were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MALDI-TOF/TOF tandem MS. In addition, we found that the levels of phosphorylation of the phaseolin changed remarkably in the seed transition from dormancy to early germination stage. Temporal changes in the extent of phosphorylation in response to physiological and metabolic variations suggest that phosphorylated phaseolin isoforms have functional significance. In particular, this prospective study supports the hypothesis that mobilization of the phaseolin in germinating seeds occurs through the degradation of highly phosphorylated isoforms. Taken together, our results indicate that post-translational phaseolin modifications through phosphorylations need to be taken into consideration for a better understanding of the molecular mechanisms underlying its regulation.     

In vivo light‐sheet microscopy resolves localisation patterns of FSD1, a superoxide dismutase with function in root development and osmoprotection

Superoxide dismutases (SODs) are enzymes detoxifying superoxide to hydrogen peroxide while temporal developmental expression and subcellular localisation are linked to their functions. Therefore, we aimed here to reveal in vivo developmental expression, subcellular, tissue‐ and organ‐specific localisation of iron superoxide dismutase 1 (FSD1) in Arabidopsis using light‐sheet and Airyscan confocal microscopy. FSD1‐GFP temporarily accumulated at the site of endosperm rupture during seed germination. In emerged roots, it showed the highest abundance in cells of the lateral root cap, columella, and endodermis/cortex initials. The largest subcellular pool of FSD1‐GFP was localised in the plastid stroma, while it was also located in the nuclei and cytosol. The majority of the nuclear FSD1‐GFP is immobile as revealed by fluorescence recovery after photobleaching. We found that fsd1 knockout mutants exhibit reduced lateral root number and this phenotype was reverted by genetic complementation. Mutant analysis also revealed a requirement for FSD1 in seed germination during salt stress. Salt stress tolerance was coupled with the accumulation of FSD1‐GFP in Hechtian strands and superoxide removal. It is likely that the plastidic pool is required for acquiring oxidative stress tolerance in Arabidopsis. This study suggests new developmental and osmoprotective functions of SODs in plants.     

不同年龄日本落叶松人工林生物量、碳储量及养分特征

以7、17、30和40年生4个发育阶段(幼龄、中龄、近熟和成熟阶段)的日本落叶松人工林为对象,研究了林龄对生物量、碳储量和养分特征的影响.结果表明: 在单木水平上,不同发育阶段干、枝、皮、叶、根生物量和养分浓度差异显著.随年龄增加,各器官生物量呈增大趋势,N、P、K浓度呈下降趋势,Mg浓度先降后升,Ca浓度持续升高.优势木、平均木和劣势木的各器官生物量之间差异显著,但养分浓度差异不显著,表明竞争对各器官养分浓度影响不大.在林分水平上,总生物量、碳储量和养分储量随林龄增加呈增大趋势,与幼龄林相比,成熟林分别增加217.9%、218.4%和56.4%,表明日本落叶松林生长后期能以较少的养分生产较多的干物质,养分利用效率较高.5种元素的积累量除P和K在近熟林(30年生)略有降低外,其他元素都随林龄增加而增加.N集中在叶中,Ca集中在树干,K和Mg主要集中在根,P在不同器官中的分配较均匀.日本落叶松林分年均生物量积累率、固碳率和养分积累率均随林龄的增加而降低,从幼龄林每年7.16 t·hm^-2、3.40 t·hm^-2、104.64 kg·hm^-2降低到成熟林的3.99 t·hm^-2、1.89 t·hm^-2、28.64 kg·hm^-2,表明日本落叶松林幼、中龄阶段固碳潜力大,但养分消耗也高.     

Long-distance stress and developmental signals associated with abscisic acid signaling in environmental responses

Flowering plants consist of highly differentiated organs, including roots, leaves, shoots and flowers, which have specific roles: root system for water and nutrient uptake, leaves for photosynthesis and gas exchange and reproductive organs for seed production. The communication between organs through the vascular system, by which water, nutrient and signaling molecules are transported, is essential for coordinated growth and development of the whole plant, particularly under adverse conditions. Here, we highlight recent progress in understanding how signaling pathways of plant hormones are associated with long-distance stress and developmental signals, with particular focus on environmental stress responses. In addition to the root-to-shoot peptide signal that induces abscisic acid accumulation in leaves under drought stress conditions, we summarize the diverse stress-responsive peptide signals reported to date to play a role in environmental responses.     

Spatio-temporal accumulation and activity of calcium-dependent protein kinases during embryogenesis, seed development, and germination in sandalwood

Western-blot analysis and protein kinase assays identified two Ca(2+)-dependent protein kinases (CDPKs) of 55 to 60 kD in soluble protein extracts of embryogenic cultures of sandalwood (Santalum album L.). However, these sandalwood CDPKs (swCDPKs) were absent in plantlets regenerated from somatic embryos. swCDPKs exhibited differential expression (monitored at the level of the protein) and activity in different developmental stages. Zygotic embryos, seedlings, and endosperm showed high accumulation of swCDPK, but the enzyme was not detected in the soluble proteins of shoots and flowers. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages; the enzyme was localized strongly in the storage bodies of the endosperm cells. The study also reports for the first time to our knowledge a post-translational inhibition/inactivation of swCDPK in zygotic embryos during seed dormancy and early stages of germination. The temporal expression of swCDPK during somatic/zygotic embryogenesis, seed maturation, and germination suggests involvement of the enzyme in these developmental processes.