Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.

Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.

Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.

Date of Award

5-2012

Document Type

Campus Access

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Plant and Soil Sciences

First Advisor

Om Parkash Dhankher

Second Advisor

John Burand

Third Advisor

Samuel P. Hazen

Subject Categories

Molecular Biology | Plant Biology | Plant Sciences

Abstract

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 ( 10 B) 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 andOsPIP2;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.

Share

COinS