Amino acid permease RcAAP1 increases the uptake and phloem translocation of an L-valine-phenazine-1-carboxylic acid conjugate

Abstract

Amino acid conjugates of pesticides can promote the phloem translocation of parent ingredients, allowing for the reduction of usage, and decreased environmental pollution. Plant transporters play important roles in the uptake and phloem translocation of such amino acid-pesticide conjugates such as L-Val-PCA (L-valine-phenazine-1-carboxylic acid conjugate). However, the effects of an amino acid permease, RcAAP1, on the uptake and phloem mobility of L-Val-PCA are still unclear. Here, the relative expression levels of RcAAP1 were found to be up-regulated 2.7-fold and 2.2-fold by the qRT-PCR after L-Val-PCA treatments of Ricinus cotyledons for 1 h and 3 h, respectively. Subsequently, expression of RcAAP1 in yeast cells increased the L-Val-PCA uptake (0.36 μmol/10⁷ cells), which was 2.1-fold higher than the control (0.17 μmol/10⁷ cells). Pfam analysis suggested RcAAP1 with its 11 transmembrane domains belongs to the amino acid transporter family. Phylogenetic analysis found RcAAP1 to be strongly similar to AAP3 in nine other species. Subcellular localization showed that fusion RcAAP1-eGFP proteins were observed in the plasma membrane of mesophyll cells and phloem cells. Furthermore, overexpression of RcAAP1 for 72 h significantly increased the phloem mobility of L-Val-PCA in Ricinus seedlings, and phloem sap concentration of the conjugate was 1.8-fold higher than the control. Our study suggested that RcAAP1 as carrier was involved in the uptake and phloem translocation of L-Val-PCA, which could lay foundation for the utilization of amino acids and further development of vectorized agrochemicals.

Keywords: amino acid-pesticide conjugate; overexpression; phloem mobility; subcellular localization; uptake; vacuum agroinfiltration.

Figure 1

Identification of the effects of RcAAP1 on L-Val-PCA uptake. (A) The relative expression levels of RcAAP1 at 1 h, 3 h, and 6 h after 100 μM PCA, 100 μM L-Val or 100 μM L-Val-PCA treatment of seedling cotyledons. “CK” stands for solvent treatments (0.5% dimethyl sulfoxide, 20 mM MES, 0.25 mM MgCl₂, 0.5 mM CaCl₂, pH 5.6). The reference gene RcActin was used for normalization of expression. The relative expression level represents fold-change. The experiment used three biological replicates. Asterisks on the graphs indicate statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001). (B) Growth of S. cerevisiae W303-1A carrying pYES2-RcAAP1 with a density of OD_600nm = 0.01 on SD-Gal (2% galactose) media containing 1 mM or 1.5 mM L-Val-PCA. The medium containing 0.5% DMSO was used as the control. (C) The uptake amount of L-Val-PCA in yeast cells. Transformant carrying empty plasmid pYES2 was set up as the control. The standard curve (y = 0.13052x + 0.72556) was used for HPLC determination of L-Val-PCA. The correlation coefficient was 0.99999. The experiment was replicated three times and different independent samples were used during these biological replicates. Asterisks on the graphs indicate statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2

Bioinformatic and phylogenetic analyses of RcAAP1 after submission of amino acid sequence to the databases of interPro, NCBI, SMART, and Phyre2. (A) Pfam analysis. (B) Phylogenetic analysis of RcAAP1 with homologous AAT sequences from nine species, namely Durio zibethinus, Gossypium arboretum, Gossypium hirsutum, Jatropha curcas, Mercurialis annua, Populus alba, Populus euphratica, Populus trichocarpa, and R. communis. This tree was constructed using MEGA11 with the Neighbor-Joining (NJ) algorithm. The optimal trees were statistically evaluated by bootstrap analysis with 1000 replications and the bootstrap percentages are shown at the appropriate branches. (C) Results of sequence alignments of amino acids between RcAAP1 and RcAAP3. (D, E) Domain prediction of RcAAP1 and RcAAP3. (F, G) Three-dimensional structures of RcAAP1 and RcAAP3. (H, I) Secondary structure analysis of RcAAP1 and RcAAP3.

Figure 3

Expression and localization observation of fusion RcAAP1-eGFP in the mesophyll cells of N. benthamiana at 400 x magnification by laser confocal microscopy. Plant binary vectors pCAMBIA1300-35S-PM-mCherry and pART27-eGFP was used to transiently expressed DsRed (Discosoma red fluorescent protein) and eGFP (enhanced green fluorescent protein), which were used as the localization marker and the control, respectively.

Figure 4

Overexpression of RcAAP1 were carried out in Ricinus seedlings for subcellular localization and phloem translocation functions of RcAAP1 toward L-Val-PCA. (A1, A2) Confirm of the expression of eGFP and fusion RcAAP1-eGFP under UV light at 72 h after transformation of empty vector pART27-eGFP and recombinants pART27-RcAAP1-eGFP into Ricinus seedlings by vacuum agroinfiltration. (B1, B2) Cotyledon observation of eGFP and RcAAP1-eGFP at 100 x magnification by laser confocal microscopy. (C1, C2) Cotyledon observation of eGFP and RcAAP1-eGFP at 400 x magnification. (D1, D2) Phloem observation of eGFP and RcAAP1-eGFP at 200 x magnification by laser confocal microscopy. (E1, E2) The enlargements of phloem cells that overexpressed eGFP gene and RcAAP1 gene, respectively. (F) qPCR analysis of the relative expression levels of RcAAP1 after overexpression of RcAAP1 for 72 h. (G) HPLC detection of the phloem sap concentrations of L-Val-PCA after overexpression of RcAAP1 for 72 h. The experiment was replicated three times and different independent samples were used during these biological replicates. Different asterisks on the graphs indicate statistically significant differences (**P < 0.01).