The researchers from UConn, Purdue University and Pennsylvania State University determined that one of three proton pumps found within plant cells, previously believed to have an extremely limited function, plays a critical role in plant root and shoot system growth and development by controlling cell division, expansion and hormone transport. Over-expressing the single gene that encodes this particular proton pump significantly enhances the transportation of the primary plant growth hormone, auxin, and results in plants with stronger, more extensive root systems and as much as 60 percent more foliage, the researchers report in the Oct. 7 issue of the prestigious journal Science.
"This discovery has the potential to revolutionize agriculture worldwide," said Gaxiola, an assistant professor-in-residence in UConn's plant science department. "This over-expression regulates the development of one of the most important parts of the plant, the roots. A plant with larger roots is a healthier and more productive plant, because, with a larger root system, the plant is able to get water and nutrients from larger soil areas.
"Biology textbooks tell you there are three pumps inside a plant's cell but one is less important. Our research shows that is not the case," Gaxiola said. "As it turns out, that tiny pump is required to shuttle the master pump, the plant's major engine, to the plasma membrane. That, in turn, allows the master pump to facilitate the transport of more of the growth hormone, auxin, through the plant's plasma membrane and through the plant's root and shoot systems, resulting in enhanced cell division and growth."
All plants contain three proton pumps – a master pump, known as the P-type H+-ATPase, that facilitates transport of nutrients in and out of plant cells, and two other pumps that work within plant cells. Biologists have shown that only the P-type H+-ATPase pumps protons into the space outside the cell to create changes that drive the transport of small molecules in and out of cells. Until now, they believed the AVP1 H+-PPase that Gaxiola's group over-expressed merely controlled pH levels within plant vacuoles, or large storage areas inside plant cells, and served primarily as a back-up pump to a larger vacuolar pump known as V-ATPase. Scientists believed that the larger vacuolar pump was the only one to help shuttle the master pump to and from the plant cell's plasma membrane.
In collaboration with scientists at the Massachusetts Institute of Technology and Harvard University, Gaxiola previously had created plants in which the AVP1 gene was over-expressed using the research plant Arabidopsis thaliana. As Gaxiola predicted, these plants were salt- and drought-resistant and sequestered more salt ions in their vacuoles. Surprisingly the plants also had abnormally large root and shoot systems.
Simon Gilroy, a Pennsylvania State University cell biologist, provided another piece to the puzzle when he discovered that the pH, which indicates proton concentration, was unchanged inside the cells. But the extra-cellular pH was lower, meaning it was more acidic and had a higher proton concentration.
The next clue came from plant cell biologist Angus Murphy and his colleagues at Purdue University.
"When Simon reported the acidity and the proton gradient was increased between the inside and outside of plant cells in Roberto's over expression lines, we saw an opportunity to test the model that had been used to explain the transport of the plant hormone auxin for the last 30 years," Murphy said. "This model predicts that an increased proton gradient should result in a faster rate of auxin transport. This theory never had been tested directly tested in plants where the proton gradient had been manipulated by molecular genetic techniques. When we determined that the rate of transport was increased, but the overall auxin content was not, the auxin transport model was validated."
They determined AVP1's critical role by comparing the transgenic plants to both ordinary Arabidopsis plants and mutant versions of the plant that were devoid of AVP1. They discovered that the AVP1 mutants didn't develop functional root systems and their shoots were tiny and deformed.
Gaxiola specializes in manipulating plant proton pumps for crop improvement and relied on Murphy and Purdue colleague Wendy Peer, for expertise in auxin transport in plants, and Gilroy for expertise in plant cell biology with an emphasis on roots.
Additional authors are UConn doctoral students Jisheng Li, Haibing Yang, Soledad Undurraga and Mariya Khodakovskaya; Purdue doctoral students Joshua Blakeslee, Anindita Bandyopadhyay, Boosaree Tiapiwantakun, Elizabeth Richards; Penn State doctoral student Gregory Richter; and University of South Carolina Biology Professor Beth Krizek.
Gaxiola said that early experiments to duplicate the Arabidopsis results in other crops, such as tomatoes, rice, cotton and poplar trees, indicate the team's discovery could have implications for increasing the world's food production and aiding global reforestation efforts. He predicts that within the next five years there will be a "boom" of crops genetically engineered using his team's approach. The research team's findings are likely to be particularly significant for farmers in developing countries, including Gaxiola's native Mexico, because many live in arid regions and lack irrigation systems and money for the amount of expensive fertilizers needed to feed plants with less expansive root systems.
U.S. patents currently are pending and a research licensing agreement with an international company has been signed.
For a copy of the embargoed Science paper, please contact the American Association for the Advancement of Sciences Office of Public Programs at (202) 326-6440 or scipak@aaas.org.
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Science