Subcellular Localization of the D27 Protein in Sugarcane (Saccharum spp. Hybrids) Using an Optimized Protoplast Isolation, Purification, and Transient Gene Expression Protocol

Wu, Zhuandi; Hu, Xueli; FengGang, Zan; Pan, Yong‐Bao; Burner, David; Luo, Zhengying; Liu, Jiayong; Zhao, Liping; Yao, Li; Zhao, Yong; Liu, Xin-long; Xia, Hongming; Yang, Kun; Zhao, Jun; PeiFang, Zhao; Qin, Wei; Chen, Xuekuan; CaiWen, Wu

doi:10.1007/s12355-020-00879-y

Cited by 6 publications

(2 citation statements)

References 33 publications

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“…The enzymolysis time required for different plant materials varies. During the process of sugarcane protoplast preparation, Wu et al [ 51 ] showed a trend of first increasing and then decreasing protoplast yield and viability with increasing enzymolysis time. Adjei et al [ 52 ] found that the optimal enzymolysis time for mango protoplasts was 12 h, while Wang et al [ 53 ] used 2 h for separation of passion fruit protoplasts.…”

Section: Discussionmentioning

confidence: 99%

Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328)

Yang,

Sun,

Sun

et al. 2024

Plant Methods

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Background Populus simonii × P. nigra is an ideal material for studying the molecular mechanisms of woody plants. In recent years, research on Populus simonii × P. nigra has increasingly focused on the application of transgenic technology to improve salt tolerance. However, the rapid characterization of gene functions has been hampered by the long growth cycle and exceedingly poor transformation efficiency. Protoplasts are an important tool for plant gene engineering, which can assist with challenging genetic transformation and the protracted growth cycle of Populus simonii × P. nigra. This study established an optimized system for the preparation and transformation of protoplasts from Populus simonii × P. nigra leaves, making genetic research on Populus simonii × P. nigra faster and more convenient. Major Latex Protein (MLP) family genes play a crucial role in plant salt stress response. In the previous study, we discovered that PsnMLP328 can be induced by salt treatment, which suggested that this gene may be involved in response to salt stress. Protein localization is a suggestion for its function. Therefore, we conducted subcellular localization analysis using protoplasts of Populus simonii × P. nigra to study the function of the PsnMLP328 gene preliminarily. Results This study established an optimized system for the preparation and transformation of Populus simonii × P. nigra protoplasts. The research results indicate that the optimal separation scheme for the protoplasts of Populus simonii × P. nigra leaves included 2.5% cellulase R-10, 0.6% macerozyme R-10, 0.3% pectolyase Y-23, and 0.8 M mannitol. After enzymatic digestion for 5 h, the yield of obtained protoplasts could reach up to 2 × 107 protoplasts/gFW, with a high viability of 98%. We carried out the subcellular localization analysis based on the optimized transient transformation system, and the results indicated that the MLP328 protein is localized in the nucleus and cytoplasm; thereby proving the effectiveness of the transformation system. Conclusion In summary, this study successfully established an efficient system for preparing and transforming leaf protoplasts of Populus simonii × P. nigra, laying the foundation for future research on gene function and expression of Populus simonii × P. nigra.

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Section: Discussionmentioning

confidence: 99%

Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328)

Yang,

Sun,

Sun

et al. 2024

Plant Methods

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“…The possible reason is that during enzymolysis, because of the unstable cellular osmotic environment, while the cytoplasm is fragile and unevenly concentrated, the protoplasm breaks easily, affecting its ability to divide causes difficulty in regeneration of protoplasts (Z. Wu et al, 2021).…”

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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Embryogenic callus induction, proliferation, protoplast isolation, and PEG induced fusion in Camellia oleifera

He,

Xu,

et al. 2024

Plant Cell Tiss Organ Cult

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Subcellular Localization of the D27 Protein in Sugarcane (Saccharum spp. Hybrids) Using an Optimized Protoplast Isolation, Purification, and Transient Gene Expression Protocol

Cited by 6 publications

References 33 publications

Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328)

Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328)

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

Embryogenic callus induction, proliferation, protoplast isolation, and PEG induced fusion in Camellia oleifera

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