This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.ABSTRACTWe propose to evaluate the absorption of calcium from 2 types of carrots: control carrots (regular carrots like would be purchased at a grocery store) and super carrots (genetically modified to be enriched with calcium). These studies will be performed in adults (21-30.0 yrs) of both genders who are selected to match the ethnic distribution of the Greater Houston area. A triple isotope method will be used to assess the absorption of calcium from the 2 types of carrots, and to compare that absorption with the calcium absorption from milk. Subjects will be given a meal in which the carrots have been intrinsically labeled with a stable isotope and a comparable amount of calcium via milk which has separately been labeled. In this case, 42Ca will be used to label the carrots and 48Ca used to label the milk. Each subject will be given breakfast which has a total of 250 mg of calcium. This calcium will consist of the calcium in the carrots, calcium in the milk and additional dietary calcium through the meal to reach about 250 mg. They will be given 46Ca intravenously and the relative 24 hour urinary recovery of the oral compared to the intravenous tracer used to assess calcium absorption. Our findings will lead to new sources of dietary calcium using vegetables that adults typically consume.HYPOTHESIS Hypothesis: Calcium absorption will be greater from carrots which express high levels of the plant endomembrane calcium transporter than from control carrots. The expression of CAX1 in the transgenic plants will increase total calcium, but not alter the fractional absorption of calcium.
SPECIFIC AIMS Rationale: Ultimate demonstration of our hypothesis regarding nutritional benefits to alterations in calcium partitioning in plants require human studies. The use of stable isotopes makes this feasible and safe as a method of determining a net gain in total calcium absorbed.BACKGROUND AND SIGNIFICANCE Initially, plant Ca2+/H+ antiporter genes were cloned by their ability to suppress the Ca2+ hypersensitive phenotype of a Saccharomyces cerevisiae mutant (Hirschi et al., 1996; Ueoka-Nakanishi et al., 2000). These genes are termed cation exchangers (CAX). CAX1 from Arabidopsis is a high capacity Ca2+ transporter (Hirschi et al., 1996). The properties of the CAX1 gene product indicate that it is the high capacity transporter responsible for maintaining low cytosolic-free Ca2+ concentrations in plant cells. In vitro measurements demonstrate that expression of CAX1 results in high Ca2+ uptake into the plant vacuole. The CAX1 cDNA that has been expressed in various plants was originally cloned through a yeast (Saccharomyces cerevisiae) suppression screen (Hirschi et al., 1996). The original CAX1 is a partial-length cDNA that is deregulated for H+/Ca2+ antiport and is now termed N-terminally truncated CAX 1 (sCAX1). It is this activated form of CAX1 that when expressed in plants results in increased Ca2+ accumulation (Hirschi, 1999; Park et al., 2004, 2005).This result suggests that ectopic deregulated CAX1 expression could significantly increase the Ca levels of edible roots. We have chosen to use carrots as the vegetables in which we ectopically express deregulated CAX1. Carrots are a favorite raw vegetable snack among U.S. consumers and the popularity of carrots is rising in Japan, Canada and Mexico (Economic Research Service/USDA 1997). Carrots also contain high levels of other beneficial phytochemicals, mainly beta carotene. This increase in Ca2+ may allow us to simultaneously deliver more Ca2+ and beta carotene to consumers as value-added traits. The transgenic carrots with increased calcium levels contain two additional proteins. We have documented that each gene is a single copy in the genome. The first gene, which is also found in the controls, is the standard antibiotic resistance gene kanamycin. The nptII gene which encodes kanamycin resistance has been consumed by humans for thousands of years, as many bacteria from the soil contain this antibiotic resistance. The second gene is a plant membrane transporter which pumps more calcium into intracellular organelles. This gene is from another plant, the small weed Arabidopsis thaliana. Although Arabidopsis does not taste good, none of the proteins within the genome have been shown to be detrimental when consumed.In addition, our modified carrots have been fed to mice, and no adverse affects have been documented. Although we have not tested for allergic reactions to this membrane protein, we believe that all plants contain transporters similar to this calcium transporter. The reason for using this Arabidopsis transporter is that we have carefully defined the biochemical properties of this protein and we have cloned this into the proper expression vectors. The transgenic lines also contain segments of DNA of bacterial origin which were initially a component of the plant expression vector. These regions of DNA do not code for any proteins and are found in various foods at local grocery stores. Like kanamycin, these regions of DNA have been consumed by humans for thousands of years in doses similar to those found in these modified carrots.

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