Progress 10/01/04 to 09/30/06

Outputs
In worldwide agricultural production, phosphorus (P) is second only to nitrogen as the most limiting macronutrient. In soils, orthophosphate (Pi), the assimilated form of phosphorus, exists primarily in insoluble calcium salts or iron/aluminum oxide complexes that are inaccessible to plants. Where aggressive fertilization is employed to alleviate available Pi deficiency, runoff from agricultural lands represents a serious threat to aquatic and marine environments. In response to limiting Pi availability, plant metabolic and developmental processes are altered to enhance Pi uptake. For example, in Arabidopsis, coordinate induction of >600 genes is seen under conditions of Pi-deprivation. Perhaps the most obvious consequence of altered gene expression in Pi-deprived plants is the expansion of their root architectures and resultant increases in absorptive surface area. Pi-deprived roots exhibit transition of the primary root to determinate growth, greater frequency of lateral root formation, and increased recruitment of trichoblasts to form root hairs. Another adaptation to low soil Pi is rhizosphere acidification resulting from enhanced plasma membrane H+-ATPase activity in roots. Increased H+ extrusion results in increased displacement of Pi from insoluble soil complexes. The advantage of these adaptations to low Pi conditions is evident in the apparent universality of such responses in plants that prosper in low Pi soils. Recently we reported that overexpression of the Arabidopsis endomembrane pyrophosphatase AVP1 resulted in increased root proliferation and apoplast acidification, suggesting a mechanism that could be manipulated to produce plants that exhibit increased resilience to Pi deficiency. Moreover, the discovery that Arabidopsis and tomato plants overexpressing AVP1 are resistant to water deficit stress further enhances the potential value of this approach, as low Pi soils are prevalent in developing nations where water deficits are not easily ameliorated by irrigation. In this research we studied the expression of AVP1 in response to Pi -deprivation and characterized the improved performance of Arabidopsis, tomato, and rice plants overexpressing AVP1 (AVP1OX) under conditions of Pi stress. Plants challenged by limiting phosphorus undergo dramatic morphological and architectural changes in their root systems in order to increase their absorptive surface area. We report here that phosphorus deficiency results in increased expression of the type I H+-PPase AVP1, subsequent increased P-ATPase-mediated rhizosphere acidification, and root proliferation. Molecular genetic manipulation of AVP1 expression in Arabidopsis, tomato, and rice results in plants that outperform controls when challenged with limited phosphorus. Thus, overexpression of type I H+-PPases appears to be a generally applicable technology to help alleviate agricultural losses in low-phosphorus tropical/subtropical soils and to reduce phosphorus runoff pollution of aquatic and marine environments resulting from fertilizer application.

Impacts
We report that phosphorus deficiency results in increased expression of the type I H+-pyrophosphatase AVP1, subsequent increased P-ATPase mediated rhizosphere acidification, and root proliferation. Furthermore, molecular genetic manipulation of AVP1 expression in Arabidopsis, tomato, and rice results in plants that outperform controls when challenged with limited phosphorus. In worldwide agricultural production, phosphorus (P) is second only to nitrogen as the most limiting macronutrient. The anthropocentric approach to address this limitation has been aggressive fertilization that unfortunately represents a serious threat to aquatic and marine environments due to P runoff from agricultural lands. The availability of crops with increased root absorptive surface area will help to alleviate agricultural losses and to prevent ecological damages. In the work presented here, transgenic rice plants engineered with the AVP1 gene showed a two fold enhancement in grain yield when grown under conditions that mimic everyday agricultural practices, suggesting that this technology can potentially improve yields of this staple crop, even in marginal soils.

Publications

• No publications reported this period

Progress 01/01/05 to 12/31/05

Outputs
Plants undergo dramatic morphological and architectural changes in their root system in order to increase their absorptive surface area in response to limiting Pi; these changes include increased extension rates of root growth, an enhanced frequency of lateral root formation and a higher recruitment of atrichoblasts to form root hairs (Poirier and Bucher, 2002, Abel et al., 2002). Furthermore, the literature shows that white lupin, a plant with a very efficient Pi scavenging capacity, up-regulates the abundance and activity of the P-ATPase as a response to limiting Pi (Yan et al., 2002). The up-regulation of the P-ATPase together with the enhanced production of lateral roots and larger and denser root hairs are essential for the rhizosphere acidification capacity displayed by the proteoid roots of these plants in response to soil Pi deficiency (Yan et al., 2002). Other studies have shown that Arabidopsis thaliana plants grown in low Pi develop root systems with higher numbers of lateral roots and larger root hairs (Lopez-Bucio et al., 2002). Of note, these Pi-starved Arabidopsis plants are more sensitive to auxin (Lopez-Bucio et al., 2002). Interestingly, this enhanced auxin sensitivity can be associated with the modifications in root architecture. As mentioned earlier the up-regulation of the vacuolar H+-PPase has been documented in Brassica napus (rapeseed) suspension cultures grown under limiting Pi (Palma et al., 2000). Furthermore, we have recently shown that in Arabidopsis, the type I H+-PPase AVP1 controls auxin transport and consequently auxin-dependent development. Significantly, changes in the expression of AVP1 affect the abundance and activity of the P-ATPase (Li et al., 2005). Based on these results, we propose the following working model for the role of AVP1 in plant response to low Pi 1) Low Pi enhances AVP1 expression. 2) Higher AVP1 facilitates P-ATPase vesicle trafficking to the plasma membrane. 3) Higher P-ATPase at the plasma membrane results in a more acidic apoplast and/or rhizosphere that facilitates both the uptake of Pi and the influx of auxin. Of note, it has been shown that intracellular auxin induces the transcription of the P-ATPase establishing a positive feedback loop. This model provides a link between enhanced auxin sensitivity, root architecture modifications, and rhizosphere acidification triggered by Pi deficiency. The unifying link could be the type I H+-PPase AVP1. In order to challenge this model we are postulating the following testable predictions regarding the cellular events triggered by limiting Pi conditions in Arabidopsis root systems. 1. AVP1 induction precedes P-ATPase induction. 2. AVP1 inhibition prevents P-ATPase induction. 3. AVP1 expression is required for lateral root development. 4. AVP1 expression is under the regulation of auxin. 5. The mechanism of up-regulation of the P-ATPase mediated by AVP1 occurs naturally in some Arabidopsis ecotypes. 6. Plants engineered to overexpress AVP1 are expected to outperform control plants under limiting Pi conditions.

Impacts
Phosphorus is a non-renewable resource that has been unevenly distributed in agricultural soils of the world. In developed countries too generous applications of P fertilizers pose an ecological threat due to runoff losses that induce alga blooms in ponds and rivers. In sharp contrast, the availability of P in the soils of developing countries has been increasingly depleted. The plants can use only 10-30% of the applied P-fertilizers, and the remaining P becomes unavailable for the majority of our current crops. Therefore, the generation of new plants with an improved P uptake capacity should have a positive impact in both developed and developing countries. Our studies on the characterization of the Arabidopsis H+-PPase AVP1 suggested that an effective way to engineer root development is by the up-regulation of AVP1 (Li et al, Science, 2005; Park et al, PNAAS, 2005). In this study we will evaluate if the root architecture changes triggered by AVP1 up-regulation enhance the capacity of Arabidopsis plants to grow under Pi-limitations. If indeed the up-regulation of this H+-PPase results in an enhanced plant growth capacity under limiting Pi conditions, the possibilities of generating Pi-uptake efficient transgenic crops will be open with obvious implications for agriculture and ecology in both developed and developing countries. We will also test our hypothesis that places AVP1 in the cross roads between the developmental plant responses to low Pi and auxin sensitivity

Publications

• No publications reported this period

Progress 01/01/04 to 12/31/04

Outputs
Earlier work has shown that up-regulation of the Arabidopsis H+-PPase (AVP1) resulted in plants with a remarkable tolerance to abiotic stress. A further characterization of these AVP1 overexpressing plants (AVP1OX) showed that they developed a much larger root system than controls when grown hydroponically. To determine if the enhanced root systems that result from AVP1 up-regulation would be instrumental for growth under limiting Pi conditions, we challenge control and two AVP1OX lines (AVP1-1 and AVP1-2) to germinate and grow in a natural low-Pi (1 ppm) soil. The growth of all plants was delayed as compared to normal conditions. However, the AVP1OX plants developed greater leaf areas than their Col-0 counterparts at different stages of growth. When scored at day 50 post-germination, the AVP1-1 line had 6-fold greater leaf area than controls, while the AVP1-2 plants showed a 2-fold increase. At day 90 post germination, all plants gained in leaf area, but the AVP1OX plants were proportionately larger the Col-0 controls. These results indicated that up-regulation of the H+-PPase AVP1 enhances the performance of plants in real life conditions. We performed similar experiments in artificial nutrient media to learn more about the anatomical and physiological alterations involved in this AVP1-mediated growth advantage in a low-Pi soil. Plants, as non-motile organisms, depend on their developmental plasticity to respond appropriately to environmental cues. For example, nutrient availability has profound effects on the growth and development of plant root systems. Larger root systems provide greater root-media contact allowing more efficient uptake of limiting nutrients; this is particularly important for the uptake of Pi. AVP1OX seedlings developed more robust root systems than Col-0 when seeds were germinated and seedlings grown for 12 days in the presence of low Pi (10 microM). The AVP1-1 and AVP1-2 plants developed an average of 1.5 and 2.0 cm longer primary roots than controls, respectively and developed an average of 6 and 8 lateral roots more than controls, respectively. To further document the differences observed, seedlings from control and AVP1OX plants where germinated and allowed to grow for 20 days in Pi-sufficient and Pi-deficient media. The biomass of their shoot and root systems was determined together with the total Pi-content per plant. When grown under Pi-sufficient conditions the root fresh weight of the AVP1OX plants was 56% (AVP1-1) and 69% (AVP1-2) higher than in controls. This difference was increased when the plants were grown under Pi-deficient conditions (i.e., 58% and 70% higher in AVP1-1 and AVP1-2, respectively). The shoots of the AVP1OX seedlings accumulated more biomass than control seedlings in both Pi-sufficient and Pi-deficient media. Of note, the root/shoot ratios of AVP1OX plants were 34% (AVP1-1) and 39% (AVP1-2) higher than in controls under Pi-sufficient conditions. In the Pi-deficient media, the difference was maintained in the case of the AVP1-1 line (36%) but reduced in the case of the AVP1-2 line (16%).

Impacts
Phosphorus is a non-renewable resource that has been unevenly distributed in agricultural soils of the world. In developed countries generous applications of P fertilizers pose an ecological threat due to runoff losses that induce alga blooms. In sharp contrast, the availability of P in the soils of developing countries has been increasingly depleted. Of note, only 10-30% of the applied P-fertilizers can be used by the plant, the remaining P becomes unavailable for the majority of our current crops. Therefore, the generation of new crops with an improved P uptake capacity should have a positive impact in both developed and developing countries. Enhanced root systems contribute to water-use efficiency and facilitate the extraction of micro- and macronutrients from the soil. The proposed research will shed light on our understanding of the morphological and physiological adaptations that result in crops with enhanced Pi-uptake capacities. In AVP1OX plants, where the up-regulation of a single gene results in enhanced root architecture and Pi uptake ability, will certainly have a positive impact on agricultural production, helping to meet the challenge of world hunger.

Publications

• No publications reported this period