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Author ORCID Identifier

0000-0001-8432-2143

AccessType

Open Access Dissertation

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Plant Biology

Year Degree Awarded

2022

Month Degree Awarded

September

First Advisor

Om Parkash Dhankher

Second Advisor

Baoshan Xing

Third Advisor

Jason White

Fourth Advisor

Elsbeth Walker

Fifth Advisor

Michelle DaCosta

Subject Categories

Agriculture

Abstract

Plasma membrane intrinsic proteins (PIPs) are major intrinsic proteins (MIPs) with the ability to permeate water and small uncharged solutes as well as metalloids including arsenic (As), through the cell membranes. Arsenic is a highly toxic element that occurs naturally in the earth's crust or from anthropogenic activities with a severe poisonous effect on most living organisms. Rice, as the daily staple food for more than half of the world population, accumulates higher As contents than any other economic crop due to its growing conditions in flooded paddy fields. Members from the NOD26-like intrinsic proteins (NIP) subfamily of MIP were shown to be the main route for AsIII uptake in rice along with Si transport. Strategies to knock down these genes are not feasible because it comes with unfavorable yield loss (90%) as well as biotic and abiotic stress susceptibility.

Here, we characterized two members of the PIPs (OsPIP1;3 and OsPIP2;6) subfamily as a potential alternative to the NIPs for their role in As transport and accumulation in rice. The qRT-PCR analyses showed the differential expression of both PIP genes in response to arsenite (AsIII) exposure. Further, the transcript abundance analyses clearly indicated a root-specific expression of OsPIP1;3, whereas OsPIP2;6 showed more shoot-specific expression. We used the RNAi knockdown and overexpression approaches to examine the role of these two proteins in As transport in rice. Further, we developed transgenic rice plants expressing the GUS gene fused to the OsPIP2;6 promoter region to study its tissue-specific expression in rice. OsPIP2;6 expression was present in the vasculature system of both roots and shoots. Previous studies showed the root-specific expression and localization of OsPIP1;3 mainly to the endodermis of the roots.

Rice Ospip1;3 and Ospip2;6 RNAi lines showed a significant decrease in As accumulation in rice shoots with no significant difference in accumulation in root at short-term hydroponics studies. In contrast, the transgenic OsPIP1;3 and OsPIP2;6 overexpression plants showed significant increases in As accumulation in the shoots with no difference in the root as compared with WT plants. Long-term accumulation experiments of mature rice plants showed that As also decreased in shoots, flag leaves, and grains of the transgenic RNAi knockdown lines for both genes relative to WT plants.

Analysis of overexpressing OsPIP1;3 and OsPIP2;6 lines showed contrasting results to the RNAi knockdown lines. For short-term As uptake analysis in hydroponic setting in the seedlings of OE lines for both genes showed significantly higher As in shoots and no change in roots, compared to WT controls. However, the overexpression (OE) plants when grown until maturity showed different accumulation patterns. OsPIP1;3 OE plants accumulated slightly more As in roots and grains, while no significant difference in shoot and flag leaves, except line #7 which showed 105% and 23% increase As accumulation in roots and flag leaves, respectively. OsPIP2;6 mature plants exhibited increased As accumulation in shoots and flag leaves, while roots and grains were insignificantly lower than in the WT plants. These findings suggest that OsPIP1;3 and OsPIP2;6 are involved in As translocation from roots to shoots but not in As uptake from the rhizosphere into rice root. The overall set of results concludes that OsPIP1;3 and OsPIP2;6 are AsIII transporters. To our knowledge, this is the first report that validates the in-planta role of PIPs in As transport.

Additionally, as an alternative approach to transgenic approach, we studied the effect of nanoscale sulfur (NS) on rice seedlings and mature plants to alleviate the As toxicity and accumulation in rice. Application of NS increased seedling biomass and seeds yield by 40% and 26%, respectively, compared to control-treated plants. Soil amendment with NS and AsIII alleviated As toxicity and showed rice enhanced growth with 159% and 248% more shoot and root biomass, respectively, compared to non-amended plants exposed to AsIII alone. NS application also decreased As accumulation in roots and shoots of the NS+AsIII treated plants by 32% and 11%, respectively, compared to the AsIII-only treated plants. NS application positively impacted mature plants treated with AsIII, producing 76%, 110%, and 108% more dry shoot biomass, seed number, and seed yield, respectively, with lower As by 69, 38, 19, and 54% in their roots, shoots, flag leaves, and grains, respectively. These findings clearly demonstrate a significant potential for NS application as a sustainable soil amendment approach to decrease As in the food chain for food safety and simultaneously enhance crops productivity on As contaminated soils.

DOI

https://doi.org/10.7275/31073796

Available for download on Friday, September 01, 2023

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