br Materials and methods br Results br Discussion Our

Materials and methods
Results
Discussion Our previous investigation demonstrated that some genes in the latex that were involved in rubber biosynthesis were regulated by exogenous JA, and Hbvp1 is one of the JA-responsive genes in the latex of rubber trees [23]. In this study, the Hbvp1 encoding for V-PPase was cloned from the latex of H. brasileinsis and the deduced amino Fmoc-Asp(OtBu)-OH for sequence of Hbvp1 was highly homologous to the V-PPases in B. vulgaris, A. thaliana, P. communis, V. raiata and O. sativa, etc., with a conserved KAADVGADLVGKVE motif, the putative catalytic center of V-PPase [1]. In higher plants, V-PPase is a membrane-bound H+-PPase that was first observed on the vacuole. Since then, V-PPase has been discovered on other organelles, such as mitochondria [32], chloroplasts [33], the Golgi apparatus [34], and the plasma membrane [35]. In this research, we further demonstrated that Hbvp1 is located on the membrane of the rubber particle, a special organelle for rubber biosynthesis in the latex of rubber trees. These data indicated that Hbvp1, the product of the Hbvp1 gene, is a K+-dependent type I V-PPase, and the optimum concentration of K+ is 60mmolL−1 for Hbvp1 activity on the rubber particles. The main role of the rubber particle membrane-bound Hbvp1 may be to hydrolyze in situ the PPi released from rubber biosynthesis, and further to regulate rubber biosynthesis. It was proposed that rubber transferase and other related proteins or enzymes on the rubber particle might form a rubber transferase complex [15], which might be the elemental unit for rubber biosynthesis. Elucidating the molecular structure of the rubber biosynthesis-related protein complex could reveal the regulatory mechanisms of rubber biosynthesis in H. brasiliensis. Since Hbvp1 is a rubber particle membrane-associated protein and can regulate the rubber biosynthesis, it is intriguing to verify whether Hbvp1 is a component of the rubber biosynthesis-related protein complex on the rubber particle. Some V-PPase genes of plants are inducible and have essential roles in plant development and response to environmental changes [4], [36]. Stress conditions, such as cold, anoxia, chemical stimulation, wound, salt, osmotic shock and plant hormones, etc., could induce the gene expression and enhance the activity of V-PPase [37], [38], [39]. Our results demonstrated that JA could regulate the gene expression of Hbvp1 at both the transcription level and the translation level, and accordingly increase the hydrolytic activity of Hbvp1 on the rubber particles of H. brasiliensis. These data are consistent with other reports that the gene expression of V-PPase is regulated not only at the transcription level [40], but at the post-translational protein level [41]. The localization of the JA-responsive Hbvp1 on the rubber particles will facilitate investigation of the physiological function of JA in rubber trees. Our earlier studies suggest that JA might be an important molecule that regulates rubber biosynthesis in rubber trees [23], but the underlying mechanism is unknown. In this research, the antibody raised against Hbvp1 reduces the efficiency of incorporating [14C]IPP into rubber molecules during the rubber biosynthesis in vitro through interacting with Hbvp1 on the rubber particles; therefore, Hbvp1 might be one of the enzymes/proteins regulating rubber biosynthesis on the rubber particles. Taking these results together, one of the mechanisms by which JA regulates rubber biosynthesis might be through induction of the expression of Hbvp1 and increased Hbvp1 activity on the rubber particles, so that feedback inhibition by PPi released during rubber biosynthesis can be promptly reduced by Hbvp1 through the hydrolysis of PPi to Pi. Of course, it could not be excluded that other PPases in the latex could also be engaged in removal of PPi generated from the rubber biosynthesis. These results suggest that Hbvp1 might be a unique rubber particle membrane-associated V-PPase that is responsive to JA stimulation and is involving in regulation of the rubber biosynthesis of H. brasiliensis.