Up-regulation of a H+- pyrophosphatase (H+-PPase) as a strategy to engineer drought-resistance crop plants

Drought resistant is a desirable trait in agriculture during a period of climate change and water depletion. Transgenic crops over expression of the vacuolar H+-pyrophosphate (H+-PPase) AVP1 in the model plant Arabidopsis thaliana resulted in enhanced performance under soil water deficits, as studied by Park et al.(2005) AVP1 plays a large role in root development through the facilitation of auxin fluxes. Auxins are a class of plant hormones that play a role in the coordination of growth and behavioral processes and are essential for plant body development. Researchers looked to improve crop performance by expressing AVP1 in commercial tomatoes, Lycopersicon esculentum. The study resulted in an increased root biomass, greater pyrophosphate-driven cation transport into root vacuolar fractions, and enhanced recovery of plants from an episode of soil water deficit stress. Transgenic crops engineered to tolerate periods of drought could significantly increase yields for many developing countries and contribute to the fight against famine.
Park S., Li J., Pittman J., Berkowitz G., Yang H., Undurraga S., Morris J., Hirschi K., Gaxiola R. Up-regulation of a H+– pyrophosphatase (H+-PPase) as a strategy to engineer drought-resistance crop plants. 2005. PNAS 102 (52) 18830-18835

The transformation of Lycopersicon esculentum tomatoes was performed by means of the Agrobacterium-mediated transformation method using cotyledon and hypocotyl explants. The AVP1D1 gene is the gain-of-function mutant of the AVP1 gene that has a coordinated increase of both PPi hydrolytic activity and PPi-dependaent H+-translocation. Homozygous T2 XAVP1D lines were selected to use in all of the experiments reported in the study.
A Southern blot analysis indicated the absence of the transgene from control plants and the presence of the 35S AVP1D construct in genomic DNA of the transgenic tomato plants. In all instances, genomic DNA was digested with EcoR1, separated on a 0.9% agarose gel by electrophoresis, and probed with BgIII fragment of the AVP1D open reading frame. In total, five transgenic lines were generated. The relative intensities of the transgenic lines XAVP1D–1, XAVP1D–2, and XAVP1D-3 were observed through a Western blot. A 200%, 230%, and 150% level increase of H+-PPase protein in their root tonoplast membrane fractions was observed when compared to the control line.
Researchers continued on to study the chemical and transport effects of the recombinant protein in the transgenic tomato lines. The results indicated the transgenic lines have a mean 56% increase in H+-PPase hydrolytic activity when compared to the control plants. In addition, kinetics of PPi– and ATP-dependent 45Ca2+uptake into vacuolar membrane vessicles from transgenic and control plants were monitored. It was observed that the PPi-dependent 45Ca2+uptake was 31% greater in vesicles from the XAVP1D lines than the control, whereas ATP-dependent 45Ca2+uptake was unchanged by AVP1D expression.
Park et al. conducted soil water deficit experiments with seeds from T2homozygous vector-control and XAVP1D expressing lines were germinated on Murashige and Skoog (MS) inorganic salts, 3% (wt/vol) sucrose, MS vitamins, and 100 mg/liter kanamycin. Plants were grown for a period of 5 weeks in soil and watered regularly to field capacity. Then, the soil was allowed to dry by withholding water. In every experiment, stress was induced by withholding water until all plants showed severe signs of drought (i.e., visivle loss of turgor and wilting). In total, seven independent experiments were performed with vector controls and T2 XAVP1D lines. Three of them were condicted at the greenhouse facilities of College Station, Texas, and four at the Agricultural Biotechnology greenhouse of the University of Connecticut. Researchers found that the transgenic plants demonstrated recovery after relief of the water deficit stress that was not seen in the control group.
One downside of H+-PPase overexpression could be the accumulation of toxic metals in the fruit of transgenic plants. The researchers evaluated fruit contents both the control and XAVP1D plants and found no significant difference in the levels of Pb2+, Mo2+, Mn2+, Cd2+, Zn2+, Cu2+, Fe2+, or Ca2+.
Leaf water potentials of four control plants and a totally of eight transgenic plants were monitored during the imposed stress period. Researchers observed that transgenic plants maintained greater leaf water potentials and take up greater amounts of water during imposed soil water deficits. Transgenic pants maintained greater leaf water potentials from day 4 onward compared with control plants. During the end of the stress cycle, the leaf water potential of XAVP1D plants was 0.2–0.3 MPa greater than control plants at the same day of stress. It was visually evident that the XAVP1D had more enhanced plant water status during the latter part of the stress trial compared with controls at day 5 of the imposed stress.
Once possible explanation for greater leaf water potentials and enhanced plant performance could be that stomata closure was greater in XAVP1Dplants, thus restricting water usage during stress. This hypothesis was not supported by the gas exchange analysis where stomata conductance was similar in both sets of plants throughout the course of the water stress. Researchers attributed the enhanced plant performance of the transgenic crops to the significantly greater water uptake during stress periods. Water uptake in transgenic plants was significantly greater when compared with control plants between days 3 and 5 (by 14%), days 5 and 6 (by 75%), and days 6 to 8 (by 45%).
Lastly, an increased root growth in the AVP1-expressing plants could be the cause of the water deficit recovery phenotype. A visual observation and root dry weight supported the notion that transgenic tomato plants had significantly more extensive root systems.
Thus, the results of this experiment suggest that AVP1D expression increases root growth, water uptake, leaf water potentials, and plant survival under soil water deficit. Conversely, a loss of function study of the AVP1D gene supported the notion that it is crucial for plant growth and development. The results of this study have exciting implications for agriculturalist. Hopefully, this gene can be engineered into a variety of modern crops in order to improve yields under water deficit conditions. 

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