Cloning And Functional Characterization Of PgTIP1, A Tonoplast Aquaporin In Panax Ginseng

Aquaporins are water channel proteins found in the membranes and belongto the major intrinsic protein (MIP) family. The regulation of cellular watermovement and homeostasis plays an important role in plant growth anddevelopment. Differential expression of genes that encode different aquaporinisoforms during plant development has shown to be associated with variousphysiological processes. Habituation is generally defined as a stable heritableloss in the requirement of cultured plant cells for one or more externally suppliedgrowth regulators. The physiological basis of habituation is still unknown. Inthis study, we cloned a tonoplast aquaporin gene PgTIP1, when exploring themechanism of habituation in Panax ginseng, which strong differentiallyexpressed in habituated ginseng cells. Further functional characterization ofPgTIP1was also conducted.Habituation is considered an in vitro epigenetic switch to autotrophy forexternally supplied plant hormones. It has been reported in carrot, tobacco,potato, sugar beet, and soybean cultures. We established a habituated ginsengcell line by screening the auxin and cytokinin dependent ginseng cells, whichcan grow in vitro on hormone-free medium with the similar growth speedcompared with that of the hormone dependent cells. The morphologicalcharacter such as the cell shape and size of these habituated ginseng cells is alsosimilar to that of non-habituated ginseng cells. The total saponin content of thehabituated ginseng cells was higher than the non-habituated cells. Theexpression levels of genes encoding squalene synthase (SQS) and squaleneepoxidase (SQE), key enzymes of saponin biosynthesis, were also higher in thehabituated ginseng cells compared with the non-habituated cells. Our resultsindicate that the habituated ginseng cell line has a greater capacity for saponinproduction compared with that of the non-habituated ginseng cells. Since thereare side effects of exogenous growth regulators, habituation in Panax ginseng isof special significance.In order to explore the molecular mechanism of autonomous growth, wescreened the differential express genes in hormone autotrophic ginseng cell lineusing SSH (suppression subtractive hybridization) method and cloned anaquaporin gene (PgTIP1) specially expressed in hormone autotrophic cells. Using real-time reverse transcript (RT)-PCR method, the expression of PgTIP1under different hormone treatment was studied in suspension cultured hormoneautotrophic ginseng cells. It was found that the transcripts level of PgTIP1 wasup-regulated markedly by KT (0.25 mg l^-1) treatment, down-regulated by 1.5mg l^-1 2,4-D and also inhibited by treated with both of them at the same time.Previous studies provide little information about the relationship between plantaquaporins and auxin/cytokinin. And no research was reported on aquaporin andthe phenomenon of autonomous growth. For further confirmation of this newaquaporin gene in Panaxginseng, we did the bioinformatics and in vitrofunction analysis. Sequence alignment and predicted structure of PgTIP1showed its distinct characteristic of plant aquaporin and its water channelactivity was demonstrated by hetero-expressed in Xenopus oocytes system. TheGFP-tagged PgTIP1 decorated the tonoplast membrane, which showed itssubcellular localization. Since the specific expression of PgTIP1 in hormoneautotrophic ginseng cells indicated its potential involvement in cell division andgrowth, over-expression of PgTIP1 in Arabidopsis was conducted to explore itspossible physiological function. Results showed that the early growth anddevelopment speed of PgTIP1 over-expressed Arabidopsis was faster than thatof the wild type plants, which was exhibited by the longer roots, bigger leaf cellsand earlier flowering time. Moreover, both the seed size and the seed mass ofArabidopsis plants over-expressingPgTIP1were greatlyenhanced comparedwith those of the wild type. The seeds from transgenic Arabidopsis plants had1.85-fold higher fatty acid content than that of the wild type control. Theseresults indicate that PgTIP1 might have a role in substance storage andsecondary metabolism, in addition to promoting cell expansion/elongation.