Abstract
The Monopolar Spindle 1 (Mps1) protein is a dual-specificity kinase that plays a critical role in the progression of the cell cycle. Studies on biochemical changes promoted by Mps1 protein inhibition are important for the development of somatic embryogenesis in plants; however, such work has not been previously performed for Araucaria angustifolia. We analyzed the effects of Mps1 protein inhibition on differential protein regulation and the effects of such differential regulation on the carbohydrate, nitric oxide (NO) and polyamine (PA) contents in embryogenic suspension cultures of this species. A proteomic analysis by the shotgun method was performed using mass spectrometry. The PA and carbohydrate contents were determined by high-performance liquid chromatography, while NO was analyzed by fluorescence microscopy. A total of 1518 proteins were identified, and 157 and 162 proteins were down- and up-regulated, respectively, in embryogenic suspension cultures incubated with 10 µM Mps1 inhibitor. Inhibition of the AaMps1 protein affected the abundance of proteins related to cell cycle division by reducing the accumulation of both cell division control protein 48 and NAP1-related protein 2. In addition, the abundance of the sucrose synthase 3 protein was down-regulated, which reduced sucrose contents. Moreover, a significant reduction in endogenous NO and free putrescine contents was observed in the embryogenic suspension cultures treated with the Mps1 inhibitor. These results show that AaMps1 protein inhibition affects the metabolism of proteins, carbohydrates, NO and PAs, which ultimately interferes in the development of somatic embryogenesis in A. angustifolia.
Key Message
The inhibition of AaMps1 during somatic embryogenesis of Araucaria angustifolia modulates the abundance of proteins and the contents of nitric oxide, carbohydrate and polyamines.
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Abbreviations
- HSP:
-
Heat shock protein
- LEA:
-
Late embryogenesis abundant
- Mps1:
-
Monopolar spindle 1
- NO:
-
Nitric oxide
- PAs:
-
Polyamines
- PEMs:
-
Pro-embryogenic masses
- Put:
-
Putrescine
- ROS:
-
Reactive oxygen species
- Spd:
-
Spermidine
- Spm:
-
Spermine
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Acknowledgements
Funding for this work was provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (444453/2014-8) and the Fundação Carlos Chagas Filho de Amparo a Pesquisa no Estado do Rio de Janeiro (FAPERJ) (E26/010.001507/2014; E26/202.969/2016). This study was financed also by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. VPMA and RSR acknowledge the scholarship funded by FAPERJ. JCDP thanks the CAPES – Finance Code 001 for the scholarship.
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JCDP conducted the experiments, analyzed the data, and wrote the manuscript. JCDP, MALC, VS, EISF, and CSC conceived and designed the experiments and revised the manuscript. RSR, ALWS, PE and VPAM participated in the experiments and data analysis. All authors read and approved the final manuscript.
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Figure S1
Settled cell volume (SCV) of A. angustifolia embryogenic suspension cultures during 30 days of incubation with and without 10 μM Mps1 inhibitor SP600125. (Mean ± SD; n = 5) (TIFF 366 kb)
Figure S2
Venn diagram of identified proteins (a) and total protein contents (µg.g−1 FM) (b) in embryogenic suspension cultures of A. angustifolia at 15 days of incubation in the presence of different concentrations (0 or 10 µM) of Mps1 inhibitor (mean ± standard deviation; n = 3). Differentially abundant proteins in (a) were classified as up- or down-regulated by comparing the differential abundance comparing the 10 µM Mps1 inhibitor treatment with the control treatment (without Mps1 inhibitor) (TIFF 204 kb)
Figure S3
Kyoto Encyclopedia of Genes and Genomes (KEGG) map of differentially regulated proteins that are related to carbohydrate metabolism and were identified in the embryogenic suspension cultures of A. angustifolia between the 10 µM Mps1 inhibitor treatment and the control treatment (without Mps1 inhibitor) (TIFF 165 kb)
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Douétts-Peres, J.C., Aragão, V.P.M., Cruz, M.A.L. et al. AaMps1 protein inhibition regulates the protein profile, nitric oxide, carbohydrate and polyamine contents in embryogenic suspension cultures of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae). Plant Cell Tiss Organ Cult 138, 273–286 (2019). https://doi.org/10.1007/s11240-019-01623-5
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DOI: https://doi.org/10.1007/s11240-019-01623-5