Multiple amino acid transporters as carriers load L-valine-phenazine-1-carboxylic acid conjugate into Ricinus sieve tubes for the phloem translocation.

Some transporters play important roles in the uptake and acropetal xylem translocation of vectorized agrochemicals. However, it is poorly understood the basipetally phloem-loading functions of transporters toward vectorized agrochemicals. Here, L-Val-PCA (L-valine-phenazine-1-carboxylic acid conjugate) uptake was demonstrated carrier-mediated. RcAAP2, RcANT7, and RcLHT1 showed a similarly up-regulated expression pattern from 62 transporter coding genes in Ricinus at 1 h after L-Val or L-Val-PCA treatment. Subcellular localization revealed that fusion RcAAP2-eGFP, RcANT7-eGFP and RcLHT1-eGFP proteins were expressed in the plasma membrane of mesophyll and phloem cells. Yeast assays found that RcAAP2, RcANT7, and RcLHT1 facilitated L-Val-PCA uptake. To further demonstrate the phloem-loading functions, using vacuum infiltration strategy, an Agrobacterium-mediated RNA interference (RNAi) protocol was constructed in seedlings. HPLC detection indicated that L-Val-PCA phloem sap concentrations were significantly decreased 54.5 %, 27.6 %, and 41.6 % after silencing for 72 h and increased 48.3 %, 52.6 %, and 52.4 % after overexpression, respectively. In conclusion, the plasma membrane-located RcAAP2, RcANT7, and RcLHT1 can loaded L-Val-PCA into Ricinus sieve tubes for the phloem translocation, which may aid in the utilization of transporters and molecular design of phloem-mobile fungicides target root or vascular pathogens.

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

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.

Systemic fungicidal activity of phenazine-1-carboxylic acid-valine conjugate against tobacco sore shin and its translocation and accumulation in tobacco (Nicotiana tabacum L.).

BACKGROUND: Tobacco sore shin caused by Rhizoctonia solani Kühn is a major soil-borne fungal disease of tobacco, gradually causing infected stems to become thin and dry, leading to great losses to China's tobacco industry. Fungicides with phloem mobility are needed for application to foliage to effectively control root or vascular system pathogens. In this study, phenazine-1-carboxylic acid-valine conjugate (PCA-Val) with strong phloem mobility was tested for control of tobacco sore shin. In vitro fungicidal activity, systemicity, and in vivo efficacy of PCA-Val against R. solani in tobacco seedling were evaluated. RESULTS: In vitro fungicidal activity of PCA-L-Val against R. solani was lower than that of PCA or PCA-D-Val, but the in vivo protective activity and curative activity of PCA-L-Val was the highest among these chemicals tested. The systemicity tests in tobacco seedlings revealed that PCA did not possess phloem mobility, while PCA-L-Val and PCA-D-Val exhibited strong phloem mobility and could be transported and accumulated in the lower part of the seedling as well as throughout the phloem. In addition, we also found that, just like reported hormone amino acid conjugates, PCA-L-Val could be hydrolyzed by tobacco seedlings, to release free PCA. CONCLUSIONS: The current research results indicated that PCA-L-Val possess good phloem transport in tobacco and promising in vivo antifungal activity against R. solani, which can be used as a phloem-mobile fungicide against tobacco sore shin in production practice.