A New Approach for Wounding Research: MYC2 Gene Expression and Protein Stability in Wounded Arabidopsis Protoplasts

Son, Seungmin; Kwon, Miye; Im, Jong Hee

doi:10.3390/plants10081518

Cited by 7 publications

(7 citation statements)

References 51 publications

(55 reference statements)

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“…Abiotic stresses generated by enzymatic processes in sugarcane young leaves can cause continuous trauma to protoplasts. Since plants are constantly threatened by wounding throughout their lives, understanding the biological responses to wounds at the cellular level is critical ( Son et al., 2021 ). These proteins are part of a dynamic networks that change in response to enzymatic digestion.…”

Section: Discussionmentioning

confidence: 99%

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

et al. 2022

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The protoplast experimental system eis an effective tool for functional genomics and cell fusion breeding. However, the physiological and molecular mechanisms of protoplast response to enzymolysis are not clear, which has become a major obstacle to protoplast regeneration. Here, we used physiological, cytological, proteomics and gene expression analysis to compare the young leaves of sugarcane and enzymolized protoplasts. After enzymatic digestion, we obtained protoplasts with viability of > 90%. Meanwhile, the content of malondialdehyde, an oxidation product, increased in the protoplasts following enzymolysis, and the activity of antioxidant enzymes, such as peroxidase (POD), catalase (CAT), acid peroxidase (APX), and O2-, significantly decreased. Cytologic analysis results showed that, post enzymolysis, the cell membranes were perforated to different degrees, the nuclear activity was weakened, the nucleolus structure was not obvious, and the microtubules depolymerized and formed several short rod-like structures in protoplasts. In this study, a proteomics approaches was used to identify proteins of protoplasts in response to the enzymatic digestion process. GO, KEGG, and KOG enrichment analyses revealed that the abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which allow for protein biosynthesis or degradation. RT-qPCR analysis revealed that the expression of osmotic stress resistance genes, such as DREB, WRKY, MAPK4, and NAC, was upregulated, while that of key regeneration genes, such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA, and GAUT, was significantly downregulated in the protoplasts. Hierarchical clustering and identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. Our findings can facilitate the development of a standard system to produce regenerated protoplasts using molecular markers and antibody detection of enzymolysis.

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Section: Discussionmentioning

confidence: 99%

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

et al. 2022

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“…Abiotic stresses generated by enzymatic processes in sugarcane young leaves can cause continuous trauma to protoplasts. Because plants are constantly threatened by wounding throughout their lives, understanding the biological responses to wounds at the cellular level is critical (Son et al, 2021). Plant protoplasts constitute unique single-cell systems that can be subjected to genomic, proteomic, and metabolomic analysis (X.…”

Section: Discussionmentioning

confidence: 99%

An Integrated Physiological, Cytology and Proteomics Reveals Network of Sugarcane Protoplasts Responses to Enzymolysis

Zhang

Wang

Xiao

et al. 2022

Preprint

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The protoplast experimental system has been becoming a powerful tool for functional genomics and cell fusion breeding. However, the physiology and molecular mechanism during enzymolysis is not completely understood and has become a major obstacle to protoplast regeneration. Our study used physiological, cytology, iTRAQ (Isobaric Tags for Relative and Absolute Quantification) -based proteomic and RT-PCR analyses to compare the young leaves of sugarcane (ROC22) and protoplasts of more than 90% viability. We found that oxidation product MDA content increased in the protoplasts after enzymolysis and several antioxidant enzymes such as POD, CAT, APX, and O2- content significantly decreased. The cytology results showed that after enzymolysis, the cell membranes were perforated to different degrees, the nuclear activity was weakened, the nucleolus structure was not obvious, and the microtubules depolymerized and formed many short rod-like structures in protoplasts. The proteomic results showed that 1,477 differential proteins were down-regulated and 810 were up-regulated after enzymolysis of sugarcane young leaves. The GO terms, KEGG and KOG enrichment analysis revealed that differentially abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which would allow protein biosynthesis or / degradation. The RT-PCR analysis revealed the expression of osmotic stress resistance genes such as DREB, WRKY, MAPK4, and NAC were up-regulated. Meanwhile, the expression of key regeneration genes such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA and GAUT were significantly down-regulated in the protoplasts. Hierarchical clustering, identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. We used a variety of methods to figure out how young sugarcane leaves react to enzymes.

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“…Total RNA extraction and first-strand cDNA synthesis were performed as previously described [ 29 ]. qPCR was performed using gene-specific primers ( Table S1 ) and conducted on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using the SYBR Green Master Mix (Bio-Rad, Hercules, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

SNF1-Related Protein Kinase 1 Activity Represses the Canonical Translational Machinery

Son

Song

et al. 2022

Plants

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Protein biosynthesis is achieved through translation, which consumes enormous energy. Therefore, under conditions of limited energy supply, translation progress should be strictly coordinated. Sucrose non-fermenting kinase1 (SNF1)-related protein kinase 1 (SnRK1) is an evolutionarily conserved master regulator of cellular energy stress signaling in plants. Rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) SnRK1 enhance hypoxia tolerance and induce the expression of stress-related genes. However, whether SnRK1 modulates protein synthesis in plants is unknown. In this study, using translational reporter constructs transfected in Arabidopsis protoplasts we showed that the expression of OsSnRK1A and AtSnRK1.1 decreases the abundance of canonical proteins without affecting their encoding transcript levels and protein stability. Moreover, the loading of total mRNAs and GFP mRNAs into the heavy polysome fraction which is normally translated was attenuated in transgenic Arabidopsis lines constitutively expressing OsSnRK1A or AtSnRK1.1. Taken together, these results suggest that OsSnRK1A and AtSnRK1.1 suppress protein translation to maintain energy homeostasis.

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A New Approach for Wounding Research: MYC2 Gene Expression and Protein Stability in Wounded Arabidopsis Protoplasts

Cited by 7 publications

References 51 publications

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

An Integrated Physiological, Cytology and Proteomics Reveals Network of Sugarcane Protoplasts Responses to Enzymolysis

SNF1-Related Protein Kinase 1 Activity Represses the Canonical Translational Machinery

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