Subcellular localization of the D27 protein in sugarcane (Saccharum hybrids spp.) using an optimized protoplast isolation, purification, and transient gene expression protocol

Authors: WU, ZHUAN-DI - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; HU, XIN - YUNAN ACADEMY OF AGRICULTURAL SCIENCES; ZAN, FENG-GANG - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; Pan, Yong-Bao; BURNER, DAVID - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; LUO, ZHENG-YING - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; LIU, JIA-YONG - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; ZHAO, LI-PING - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; YAO, LI - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; ZHAO, YONG - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; LIU, XIN-LONG - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; XIA, HONG-MING - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; YANG, KUN - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; ZHAO, JUN - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; ZHAO, PEI-FANG - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; QIN, WEI - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; CHEN, XUE-KUAN - YUNNAN ACADEMY OF AGRICULTURE SCIENCES; WU, CAI-WEN - YUNNAN ACADEMY OF AGRICULTURE SCIENCES

Submitted to: Sugar Tech
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/31/2020
Publication Date: 10/3/2020
Citation: Wu, Z., Hu, X., Zan, F., Pan, Y.-B., Burner, D.M., Luo, Z., Liu, J., Zhao, L., Yao, L., Zhao, Y., Liu, X., Xia, H., Yang, K., Zhao, J., Zhao, P., Qin, W., Chen, X., Wu, C. 2020. Subcellular localization of the D27 protein in sugarcane (Saccharum hybrids spp.) using an optimized protoplast isolation, purification, and transient gene expression protocol. Sugar Tech. 23(2):316-325. https://doi.org/10.1007/s12355-020-00879-y.
DOI: https://doi.org/10.1007/s12355-020-00879-y

Interpretive Summary: It takes more than 10 years to breed a new and better sugarcane cultivar for sustainable sugar productiion. This is due to the extreme complexity of sugarcane genome and a lack of complete understanding of the genetic control of agronomically favorable traits. For example, strigolactones (SLs) are a group of newly identified plant hormones mainly involved in the regulation of plant shoot and root architecture and the DWARF27 (D27) gene was involved in the SL biosynthesis. The present study was set up to localize the D27 gene in sugarcane, conduct its transient PEG-mediated gene expression by developing an optimized laboratory protocol for isolating high yield and quality sugarcne protoplasts. The results showed thar a high volume of intact protoplasts (10.94 ×106 protoplasts g-1 FW) and a survival rate of > 80.0% were achieved through enzymatic hydrolysis at constant temperature of 28 ', 60-70 rpm min-1 for 8 h in a solution containg 2.0% cellulase R-10, 0.5% macerozyme R-10, 0.6% pectolyase Y-23, 20 mM 2-(N-morpholine) ethanesulfonic acid (MES), 20 mg M KCl, and 400 mM mannitol (pH 5.7). Using green fluorescence protein (GFP) as a reporter gene, the protoplasts could be transformed most efficiently with 25% PEG4000 for 25 min. The study also located the ScD27 gene signals in the chloroplasts. The optimized sugarcane protoplast isolation, purification and transient expression protocol lays a foundation for future molecular biology research in sugarcane.

Technical Abstract: Strigolactones (SLs) are a group of newly identified plant hormones mainly involved in the regulation of plant shoot and root architecture. DWARF27 (D27) was previously proven to be involved in SL biosynthesis. The objectives of this study were to develop an optimized protocol for the isolation and purification of sugarcane protoplasts (Saccharum hybrids spp.), conduct transient PEG-mediated protoplast transfection with D27, and localize the D27 protein in sugarcane protoplasts. Total yield and viability of protoplasts were optimized for enzyme combination, mannitol concentration, and duration and temperature of enzymatic hydrolysis. High production of intact protoplasts (10.94 ×106 protoplasts g-1 FW) and a survival rate of > 80.0% were achieved through enzymatic hydrolysis at constant temperature of 28 ', 60-70 rpm min-1 for 8 h in a solution containing 2.0% cellulase R-10, 0.5% macerozyme R-10, 0.6% pectolyase Y-23, 20 mM 2-(N-morpholine) ethanesulfonic acid (MES), 20 mM KCl, and 400 mM mannitol (pH 5.7). Using GFP as the reporter gene, the protoplasts were transformed most efficiently with 25% PEG4000 for 25 min and the ScD27 protein was localized in the chloroplasts. This optimized sugarcane protoplast isolation, purification and transient expression protocol lays a foundation for future molecular biology research in sugarcane.