## 7 The Biochemical, Molecular Biological Aspects of TIA Transporters  
TIAs present in _C. roseus_ tissues for many physiological functions. Catharanthine can inhibit the growth of fungal zoospores and shows insect toxicity at physiological concentrations on the surface of _C. roseus_ leaves. The complex developmental, environmental, hormonal, organ- and cell-specific cues regulate the genes involved in the biosynthesis of TIAs [77]. Protein kinase cascade- or calcium-mediated signaling is important for the regulation of methyl jasmonate-dependent and yeast elicitor-induced TIA biosynthesis in _C. roseus_ cells [79; 80; 81]. These factors-induced de novo biosynthesis and active secretion of TIAs cost lots of energy. The long-distance transport of end products or secretion of TIAs to the target sites also requires energy [64]. The vacuolar sequestration of these TIAs more likely depends on secondary transporters, such as MATE or multidrug resistance transporter [78]. The biosynthesis of intermediates as well as end-products of TIAs happens in different tissues and organs, and they are subsequently transported to the sites for the next reactions, storage, or for their physiological functions. Therefore, the efficient long-distance transport is essentially required. Usually, three basic transport or trafficking processes exist for organic compounds in plant cells, membrane vesicle trafficking of substances, protein-aided transport/subcellular trafficking and membrane transporters-mediated across-membrane transport [3; 82]. Although great attention was paid to the transport of TIAs long time ago, now it is recognized that a passive diffusion is usually impossible for transport of highly charged TIAs, likewise for an unspecific ion-trap mechanism [20; 83]. Plenty of evidence indicates that many alkaloids such as berberine in _Coptis japonica_, nicotine in tobacco, or terpenoids are transported across the plasma membrane by ABC transporters. For example, CjMDR1 expressed in the xylem of the rhizome [51] and CjABCB2 expressed in cells around the xylem of the rhizome [84] are involved in the transport of berberine to the place where berberine is synthesized; NtNUP--a plasma membrane nicotine--acts as a proton symporter of nicotine [85]; multidrug and toxic compound extrusion (MATE) transporters act as vacuolar sequestration of nicotine [86; 87]; NpPDR1--a plasma membrane pleiotropic drug resistance-type ABC transporter in _Nicotiana plumbaginifolia_--transports deterpene sclareol antifungal compound [88]. Overexpression of _CjMDR1_ in _C. roseus_ cell cultures promoted a significant uptake and accumulation of ajmalicine and tetrahydrorolastionine compared with control lines after feeding these alkaloids [89]. These results suggest that TIAs can be transported by such type of ABC transporter. Proteomic study on two independent cell lines with different TIAs metabolism reveal that some differentially expressed transporters possibly involved in the transport of TIAs, including seven ABCG proteins, three multidrug resistance pumps, two multidrug resistance-associated proteins, three soluble ABC transporters (ABCE and ABCF subfamilies) and one for each MATE and peptide transporter [90]. In _C. roseus_, TIAs are proved to be accumulated in the vacuoles by various techniques [78; 91; 92]. One study indicates that a specific proton antiport system could trap TIAs in the vacuoles of _C. roseus_ mesophyll cells. The uptake of vindoline into the isolated tonoplast vesicles was dependent on ATP and H\({}^{+}\) gradient across vacuolar membrane, since it was sharply suppressed by H\({}^{+}\) gradient dissipators but unaffected by ABC transporter inhibitor [78]. Interestingly, catharanthine and anhydrovinblastine are also incorporated into the vacuole through an H\({}^{+}\)-dependent mechanism. These indicate that secondary multidrug antiporters, such as MATE, may also be involved in the vacuolar sequestration of TIAs [12].