Abstract
BackgroundPlant plasma membrane transporters play essential roles during the translocation of vectorized agrochemicals. Therefore, transporters associated with phloem loading of vectorized agrochemicals have drawn increasing attention. As a model system, castor bean (Ricinus communis L.) has been widely used to detect the phloem mobility of agrochemicals. However, there is still a lack of an efficient protocol for the Ricinus seedling model system that can be directly used to investigate the recognition and phloem loading functions of plasmalemma transporters toward vectorized agrochemicals.ResultsHere, using vacuum infiltration strategy, we overexpressed the coding gene for enhanced green fluorescent protein (eGFP) in R. communis seedlings by Agrobacterium tumefaciens-mediated transformation system. Strong fluorescence signals were observed in leaves, demonstrating that exogenous genes can be successfully overexpressed in seedlings. Subsequently, gene expression time and vacuum infiltration parameters were optimized. Observation of fluorescence and qRT-PCR analysis showed that eGFP strength and expression level reached a peak at 72 h after overexpression in seedlings. Parameter optimization showed Agrobacterium concentration at OD600 = 1.2, and infiltration for 20 min (0.09 MPa), return to atmospheric pressure, and then infiltration for another 20 min, were the suitable transformation conditions. To test the application of vacuum agroinfiltration in directly examining the loading functions of plasma membrane transporters to vectorized agrochemicals in seedlings, two LHT (lysine/histidine transporter) genes, RcLHT1 and RcLHT7, were overexpressed. Subcellular localization showed the strong fluorescent signals of the fusion proteins RcLHT1-eGFP and RcLHT7-eGFP were observed on the cell membrane of mesophyll cells, and their relative expression levels determined by qRT-PCR were up-regulated 47- and 52-fold, respectively. Furthermore, the concentrations of l-Val-PCA (l-valine-phenazine-1-carboxylic acid conjugate) in phloem sap collected from seedling sieve tubes were significantly increased 1.9- and 2.3-fold after overexpression of RcLHT1 and RcLHT7, respectively, implying their roles in recognition and phloem loading of l-Val-PCA.ConclusionsWe successfully constructed a transient expression system in Ricinus seedlings and laid the foundation for researchers to directly investigate the loading functions of plasma membrane transporters to vectorized agrochemicals in the Ricinus system.
Highlights
Plant plasma membrane transporters play essential roles during the translocation of vectorized agrochemicals
Phloem sap collection and analysis To demonstrate the role of the two transporters in phloem loading of l-Val-PCA, the transformant carrying recombinants pART27-RcLHT1-enhanced green fluorescent protein (eGFP) and pART27RcLHT7-eGFP were cultured for 18 h in 400 mL LB medium containing 50 μg/mL spectinomycin and 20 μg/ mL rifampin
Exogenous gene was expressed in Ricinus communis seedling The fluorescence signals were surveyed 72 h after pART27-eGFP was transformed into Ricinus seedlings. eGFP signals were observed in most seedling leave under UV light, but signal strength varied
Summary
Using vacuum infiltration strategy, we overexpressed the coding gene for enhanced green fluorescent protein (eGFP) in R. communis seedlings by Agrobacterium tumefaciens-mediated transformation system. Observation of fluorescence and qRT-PCR analysis showed that eGFP strength and expression level reached a peak at 72 h after overexpression in seedlings. Subcellular localization showed the strong fluorescent signals of the fusion proteins RcLHT1-eGFP and RcLHT7-eGFP were observed on the cell membrane of mesophyll cells, and their relative expression levels determined by qRT-PCR were up-regulated 47- and 52-fold, respectively. The concentrations of l-Val-PCA (l-valine-phenazine-1-carboxylic acid conjugate) in phloem sap collected from seedling sieve tubes were significantly increased 1.9- and 2.3-fold after overexpression of RcLHT1 and RcLHT7, respectively, implying their roles in recognition and phloem loading of l-Val-PCA
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