Human society has benefited greatly from plant secondary metabolites, often utilizing a variety of compounds as dyes, food additives, and drugs. In particular, pharmaceutical development has benefited greatly from plant secondary metabolites. One example of this utility is paclitaxel, a highly substituted diterpene approved in the treatment of breast cancer, ovarian cancer, non-small cell lung cancer, and the AIDS-related Kaposi’s sarcoma. Demand of paclitaxel is likely to increase, due to the current examination of paclitaxel in numerous clinical trials against a variety of other cancers. Taxus cell culture represents a production source of paclitaxel to meet future demand. However, paclitaxel production through Taxus cell culture is often variable and low. Targeted metabolic engineering of Taxus to produce superior paclitaxel-accumulating lines is a viable strategy to address variable and low yields. To facilitate the production of genetically engineered Taxus cell lines, stable transformation is required to examine the long-term effect of gene expression in vitro. Additionally, suitable transient transformation systems are necessary to characterize novel Taxus genes related to paclitaxel accumulation. A transient particle bombardment-mediated transformation protocol was developed to introduce transgenes into Taxus cells in vitro. Additionally, agroinfiltration in Nicotiana benthamiana was examined as a system to express genes related to paclitaxel biosynthesis and lead to the accumulation of the first dedicated taxane, taxa-4(5), 11(12)-diene. In regard to stable transformation, an Agrobacterium -mediated transformation protocol was developed, though this method requires further optimization for reliability and increased transformation efficiency. These transformation technologies will aid in the creation of elite paclitaxel-accumulating Taxus cell lines.

Using reverse genetics, complementation analyses, and cell biological approaches with the moss Physcomitrella patens, I assessed the in vivo function of two actin turnover proteins: actin depolymerizing factor (ADF) and its binding partner actin interacting protein 1 (AIP1). My studies identify a single ADF and AIP1 in moss. Loss-of-function analyses reveal that ADF is essential for viability, and AIP1 is required to promote normal tip cell expansion. AIP1 and ADF are diffusely cytosolic proteins that function in a common genetic pathway to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin turnover, AIP1 knockout lines and plants silencing ADF accumulate F-actin bundles along the cortex. Quantitative analysis of time-lapse F-actin movies demonstrates that AIP1 promotes and ADF is essential for cortical F-actin dynamics. The development of a complementation assay permitted dissection of the physiological relevance of regulatory mechanisms that control ADF activity. Mutant complementation analyses reveal that phosphoregulation of ADF at a conserved, N-terminal serine is important for in vivo function. Phosphomimetic ADF mutants have severe tip growth defects, but remain viable, demonstrating that ADF is critical for tip growth. A gain-of-function ADF mutant with enhanced affinity for phosphatidylinositol 4,5-bisphosphate has minor defects in tip growth, suggesting that this phospholipid regulates ADF activity in vivo. Complementation analyses with ADF/cofilin proteins from other organisms reveal that moss ADF is functionally conserved with some, but not all ADF/cofilins. Interestingly, rescue is inversely proportional to pH-sensitivity, suggesting that pH-insensitive ADF activity is important for tip growth in moss. The complementation analysis has also facilitated the identification of two temperature-sensitive mutants in moss ADF. These temperature sensitive mutants, together with the AIP1 knockout lines, will be instrumental for identifying cellular processes in plants that require actin dynamics – an open question in plant biology.

Abiotic stresses such as drought, salt, cold, heat and exposure to toxic metals adversely affect growth and productivity of crop plants and are serious threats to agriculture. Members of Stress Associated Protein (SAP) family in rice have been shown to provide tolerance to multiple abiotic stresses. There are 18 and 14 reported members of SAP family in rice and Arabidopsis, respectively. These SAPs contain A20, AN1, or both A20/AN1 zinc finger domains at the N- or C-terminus. Some members of SAP family proteins also contain extra Cys2-His2 RING motifs on the C-terminus. We describe here the functional characterization of two novel SAP genes, AtSAP10 and AtSAP11, from Arabidopsis thaliana ecotype Columbia. AtSAP10 gene contains an A20 and AN1 zinc-finger domain at the N- and C-terminal, respectively. Arabidopsis SAP10 showed differential regulation by various abiotic stresses such as heavy metals and metalloids (Ni, Cd, Mn, Zn, and As), high and low temperatures, cold, and ABA. Overexpression of AtSAP10 in Arabidopsis conferred strong tolerance to heavy metals such as Ni, Mn, and Zn and to high temperature stress. AtSAP10 transgenic plants under these stress conditions grew green and healthy, attained several-fold more biomass, and had longer roots as compared to wild type plants. Further, while these transgenic plants accumulated significantly greater amounts of Ni and Mn in both shoots and root tissues, there was no significant difference in the accumulation of Zn. AtSAP10 promoter-GUS fusion studies revealed a root and floral organ-specific expression of AtSAP10. Overexpression of AtSAP10-GFP fusion protein showed the localization in both nucleus and cytoplasm. A second gene from AtSAP family, AtSAP11, contains two AN1 zinc finger domains at N-terminal and two C2H2 zinc finger domains at C-terminus. Arabidopsis SAP11 showed differential regulation by various abiotic stresses such as heavy metals and metalloids (As, Cd and Zn), high and low temperatures, cold, and salt. Overexpression of AtSAP11 in Arabidopsis conferred moderate tolerance to heavy metals As and Zn and slightly enhanced tolerance to drought stress. AtSAP11 overexpression plants did not accumulate significantly higher amounts arsenic in shoots or roots. AtSAP11 promoter-GUS fusion studies revealed a floral organ-specific and fruit specific expression of AtSAP11. AtSAP11-GFP fusion showed an ER like localization of the fusion protein. Thus these results showed that AtSAP10 and AtSAP11 are potentially useful candidate genes for engineering tolerance to heavy metals and to abiotic stress in cultivated plants.

Aquaporins (AQPs) are channel proteins that facilitate the transport of water and various low molecular weight solutes including metalloids. Plant aquaporins have been divided into four major subfamilies: plasma membrane intrinsic proteins (PIPs), NOD26-like intrinsic proteins (NIPs), tonoplast intrinsic proteins (TIPs), and small basic intrinsic proteins (SIPs). Various studies have shown that the transport of metalloids including arsenite, antimonite, silicon and boron in plants is facilitated by members of NIP subfamily. In this study, we provided experimental evidences showing that members of rice PIP subfamily are involved in arsenite and boron permeability. RT-PCR analysis of seven OsPIPs; OsPIP1;2, OsPIP1;3, OsPIP2;4, OsPIP2;5, OsPIP2;6, OsPIP2;7, and OsPIP2;8 showed that these genes were downregulated under arsenite toxicity in shoots and roots. Whereas, these OsPIP genes were deferentially regulated in shoots and highly induced in roots by boron toxicity. Heterologous expression in Xenopus laevis oocytes showed that OsPIP2;4, OsPIP2;6, and OsPIP2;7 significantly increased the transport of arsenite. Expression of OsPIP candidate genes in HD9 yeast strain lacking the metalloids influx and efflux systems resulted in an increased boron sensitivity and accumulation. Overexpression of two OsPIP candidates; OsPIP1;3 and OsPIP2;6 in Arabidopsis yielded enhanced arsenite and boron tolerance with higher biomass and greater root length compared to wild type plants, however there was no difference in arsenic and boron accumulation in long-term uptake assays. Short duration exposure to AsIII resulted in both active influx and efflux of As in shoots and roots, suggesting a bidirectional transport activity of OsPIPs. Whereas, short-term uptake assay of tracer B (10B) in shoots and roots demonstrated increased 10 B influx in transgenic Arabidopsis lines indicating that these OsPIPs are also involved in mediating B transport in plants. We used RNAi approach to knockdown the expression of OsPIP1;3 and OsPIP2;6 in rice. We generated RNAi lines for both genes and qRT-PCR analysis showed a significant decrease in the transcript levels for OsPIP1;3 and OsPIP2;6. These RNAi lines will be the subject of future studies. These OsPIPs genes will be highly useful in developing arsenite and boron tolerant crops for enhanced yield in the areas affected by high As and B toxicity.