Improved Yield and Photosynthate Partitioning in AVP1 Expressing Wheat (Triticum aestivum) Plants.

A fundamental factor to improve crop productivity involves the optimization of reduced carbon translocation from source to sink tissues. Here, we present data consistent with the positive effect that the expression of the Arabidopsis thaliana H+-PPase (AVP1) has on reduced carbon partitioning and yield increases in wheat. Immunohistochemical localization of H+-PPases (TaVP) in spring wheat Bobwhite L. revealed the presence of this conserved enzyme in wheat vasculature and sink tissues. Of note, immunogold imaging showed a plasma membrane localization of TaVP in sieve element- companion cell complexes of Bobwhite source leaves. These data together with the distribution patterns of a fluorescent tracer and [U14C]-sucrose are consistent with an apoplasmic phloem-loading model in wheat. Interestingly, 14C-labeling experiments provided evidence for enhanced carbon partitioning between shoots and roots, and between flag leaves and milk stage kernels in AVP1 expressing Bobwhite lines. In keeping, there is a significant yield improvement triggered by the expression of AVP1 in these lines. Green house and field grown transgenic wheat expressing AVP1 also produced higher grain yield and number of seeds per plant, and exhibited an increase in root biomass when compared to null segregants. Another agriculturally desirable phenotype showed by AVP1 Bobwhite plants is a robust establishment of seedlings.

Assessing Long-Distance Carbon Partitioning from Photosynthetic Source Leaves to Heterotrophic Sink Organs with Photoassimilated [14C]CO2.

Phloem loading and long-distance transport of photoassimilate from source leaves to sink organs are essential physiological processes that contribute to plant growth and yield. At a minimum, three steps are involved: phloem loading in source organs, transport along the phloem path, and phloem unloading in sink organs. Each of these can have variable rates contingent on the physiological state of the plant, and thereby influence the overall transport rate. In addition to these phloem transport steps, rates of photosynthesis and photosynthate movement in the pre-phloem path, as well as photosynthate utilization in post phloem tissues of sink organs also contribute to phloem transport. The protocol described here estimates carbon allocation along the entire path from initial carbon fixation to delivery to sink organs after a labeling pulse: [14C]CO2 is photoassimilated in source leaves and loading and transport of the 14C label to heterotrophic sink organs (roots) is quantified by scintillation counting. This method is flexible and can be adapted to quantify long-distance transport in many plant species.

The flip side of the Arabidopsis type I proton-pumping pyrophosphatase (AVP1): Using a transmembrane H+ gradient to synthesize pyrophosphate.

Energy partitioning and plant growth are mediated in part by a type I H+-pumping pyrophosphatase (H+-PPase). A canonical role for this transporter has been demonstrated at the tonoplast where it serves a job-sharing role with V-ATPase in vacuolar acidification. Here, we investigated whether the plant H+-PPase from Arabidopsis also functions in "reverse mode" to synthesize PPi using the transmembrane H+ gradient. Using patch-clamp recordings on Arabidopsis vacuoles, we observed inward currents upon Pi application on the cytosolic side. These currents were strongly reduced in vacuoles from two independent H+-PPase mutant lines (vhp1-1 and fugu5-1) lacking the classical PPi-induced outward currents related to H+ pumping, whereas they were significantly larger in vacuoles with engineered heightened expression of the H+-PPase. Current amplitudes related to reverse-mode H+ transport depended on the membrane potential, cytosolic Pi concentration, and magnitude of the pH gradient across the tonoplast. Of note, experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing the Arabidopsis H+-PPase (AVP1) demonstrated Pi-dependent PPi synthase activity in the presence of a pH gradient. Our work establishes that a plant H+-PPase can operate as a PPi synthase beyond its canonical role in vacuolar acidification and cytosolic PPi scavenging. We propose that the PPi synthase activity of H+-PPase contributes to a cascade of events that energize plant growth.

Alternate Modes of Photosynthate Transport in the Alternating Generations of Physcomitrella patens.

Physcomitrella patens has emerged as a model moss system to investigate the evolution of various plant characters in early land plant lineages. Yet, there is merely a disparate body of ultrastructural and physiological evidence from other mosses to draw inferences about the modes of photosynthate transport in the alternating generations of Physcomitrella. We performed a series of ultrastructural, fluorescent tracing, physiological, and immunohistochemical experiments to elucidate a coherent model of photosynthate transport in this moss. Our ultrastructural observations revealed that Physcomitrella is an endohydric moss with water-conducting and putative food-conducting cells in the gametophytic stem and leaves. Movement of fluorescent tracer 5(6)-carboxyfluorescein diacetate revealed that the mode of transport in the gametophytic generation is symplasmic and is mediated by plasmodesmata, while there is a diffusion barrier composed of transfer cells that separates the photoautotrophic gametophyte from the nutritionally dependent heterotrophic sporophyte. We posited that, analogous to what is found in apoplasmically phloem loading higher plants, the primary photosynthate sucrose, is actively imported into the transfer cells by sucrose/H+ symporters (SUTs) that are, in turn, powered by P-type ATPases, and that the transfer cells harbor an ATP-conserving Sucrose Synthase (SUS) pathway. Supporting our hypothesis was the finding that a protonophore (2,4-dinitrophenol) and a SUT-specific inhibitor (diethyl pyrocarbonate) reduced the uptake of radiolabeled sucrose into the sporangia. In situ immunolocalization of P-type ATPase, Sucrose Synthase, and Proton Pyrophosphatase - all key components of the SUS pathway - showed that these proteins were prominently localized in the transfer cells, providing further evidence consistent with our argument.

Conjecture Regarding Posttranslational Modifications to the Arabidopsis Type I Proton-Pumping Pyrophosphatase (AVP1).

Agbiotechnology uses genetic engineering to improve the output and value of crops. Altering the expression of the plant Type I Proton-pumping Pyrophosphatase (H+-PPase) has already proven to be a useful tool to enhance crop productivity. Despite the effective use of this gene in translational research, information regarding the intracellular localization and functional plasticity of the pump remain largely enigmatic. Using computer modeling several putative phosphorylation, ubiquitination and sumoylation target sites were identified that may regulate Arabidopsis H+-PPase (AVP1- Arabidopsis Vacuolar Proton-pump 1) subcellular trafficking and activity. These putative regulatory sites will direct future research that specifically addresses the partitioning and transport characteristics of this pump. We posit that fine-tuning H+-PPases activity and cellular distribution will facilitate rationale strategies for further genetic improvements in crop productivity.

Structural basis for the reversibility of proton pyrophosphatase.

Proton Pyrophosphatase (H+-PPase) is an evolutionarily conserved enzyme regarded as a bona fide vacuolar marker. However, H+-PPase also localizes at the plasma membrane of the phloem, where, evidence suggests that it functions as a Pyrophosphate Synthase and participates in phloem loading and photosynthate partitioning. We believe that this pyrophosphate synthesising function of H+-PPase is fundamentally rooted to its molecular structure, and here we postulate, on the basis of published crystal structures of membrane-bound pyrophosphatases, a plausible mechanism of pyrophosphate synthesis.

Moving On Up: H(+)-PPase Mediated Crop Improvement.

Upregulation of H(+)-PPase in diverse crop systems triggers agriculturally beneficial phenotypes including augmented stress tolerance, improved water and nutrient use efficiencies, and increased biomass and yield. We argue that further research is warranted to maximize the full potential of this simple and successful biotechnology.

Constitutive and Companion Cell-Specific Overexpression of AVP1, Encoding a Proton-Pumping Pyrophosphatase, Enhances Biomass Accumulation, Phloem Loading, and Long-Distance Transport.

Plant productivity is determined in large part by the partitioning of assimilates between the sites of production and the sites of utilization. Proton-pumping pyrophosphatases (H(+)-PPases) are shown to participate in many energetic plant processes, including general growth and biomass accumulation, CO2 fixation, nutrient acquisition, and stress responses. H(+)-PPases have a well-documented role in hydrolyzing pyrophosphate (PPi) and capturing the released energy to pump H(+) across the tonoplast and endomembranes to create proton motive force (pmf). Recently, an additional role for H(+)-PPases in phloem loading and biomass partitioning was proposed. In companion cells (CCs) of the phloem, H(+)-PPases localize to the plasma membrane rather than endomembranes, and rather than hydrolyzing PPi to create pmf, pmf is utilized to synthesize PPi. Additional PPi in the CCs promotes sucrose oxidation and ATP synthesis, which the plasma membrane P-type ATPase in turn uses to create more pmf for phloem loading of sucrose via sucrose-H(+) symporters. To test this model, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated with constitutive and CC-specific overexpression of AVP1, encoding type 1 ARABIDOPSIS VACUOLAR PYROPHOSPHATASE1. Plants with both constitutive and CC-specific overexpression accumulated more biomass in shoot and root systems. (14)C-labeling experiments showed enhanced photosynthesis, phloem loading, phloem transport, and delivery to sink organs. The results obtained with constitutive and CC-specific promoters were very similar, such that the growth enhancement mediated by AVP1 overexpression can be attributed to its role in phloem CCs. This supports the model for H(+)-PPases functioning as PPi synthases in the phloem by arguing that the increases in biomass observed with AVP1 overexpression stem from improved phloem loading and transport.

Apoplasmic loading in the rice phloem supported by the presence of sucrose synthase and plasma membrane-localized proton pyrophosphatase.

BACKGROUND AND AIMS: Although Oryza sativa (rice) is one of the most important cereal crops, the mechanism by which sucrose, the major photosynthate, is loaded into its phloem is still a matter of debate. Current opinion holds that the phloem loading pathway in rice could involve either a symplasmic or an apoplasmic route. It was hypothesized, on the basis of a complementary body of evidence from arabidopsis, which is an apoplasmic loader, that the membrane specificity of proton pyrophosphatases (H(+)-PPases; OVPs) in the sieve element-companion cell (SE-CC) complexes of rice source leaves would support the existence of either of the aforementioned phloem loading mechanisms. Additionally, it was contended that the presence of sucrose synthase in the SE-CC complexes would be consistent with an apoplasmic sucrose loading route in rice. METHODS: Conventional chemical fixation methods were used for immunohistochemical localization of H(+)-PPases and sucrose synthase in rice and arabidopsis at the light microscopy level, while ultrastructural immunogold labelling of H(+)-PPases and sucrose synthase was performed on high-pressure frozen source leaves of rice. KEY RESULTS: Using immunogold labelling, it was found that OVPs predominantly localize at the plasma membrane (PM) of the SE-CC complexes in rice source leaf minor veins, while in the root meristematic cells, OVPs preferentially localize at the vacuoles. The PM specificity of OPVs in the SE-CC complexes was deemed to support apoplasmic loading in the rice phloem. Further backing for this interpretation came from the sucrose synthase-specific immunogold labelling at the SE-CC complexes of rice source leaves. CONCLUSION: These findings are consistent with the idea that, in the same way as in arabidopsis and a majority of grasses, sucrose is actively loaded into the SE-CC complexes of rice leaves using an apoplasmic step.

Arabidopsis type I proton-pumping pyrophosphatase expresses strongly in phloem, where it is required for pyrophosphate metabolism and photosynthate partitioning.

Phloem loading is a critical process in plant physiology. The potential of regulating the translocation of photoassimilates from source to sink tissues represents an opportunity to increase crop yield. Pyrophosphate homeostasis is crucial for normal phloem function in apoplasmic loaders. The involvement of Arabidopsis (Arabidopsis thaliana) type I proton-pumping pyrophosphatase (AVP1) in phloem loading was analyzed at genetic, histochemical, and physiological levels. A transcriptional AVP1 promoter::GUS fusion revealed phloem activity in source leaves. Ubiquitous AVP1 overexpression (35S::AVP1 cassette) enhanced shoot biomass, photoassimilate production and transport, rhizosphere acidification, and expression of sugar-induced root ion transporter genes (POTASSIUM TRANSPORTER2 [KUP2], NITRATE TRANSPORTER2.1 [NRT2.1], NRT2.4, and PHOSPHATE TRANSPORTER1.4 [PHT1.4]). Phloem-specific AVP1 overexpression (Commelina Yellow Mottle Virus promoter [pCOYMV]::AVP1) elicited similar phenotypes. By contrast, phloem-specific AVP1 knockdown (pCoYMV::RNAiAVP1) resulted in stunted seedlings in sucrose-deprived medium. We also present a promoter mutant avp1-2 (SALK046492) with a 70% reduction of expression that did not show severe growth impairment. Interestingly, AVP1 protein in this mutant is prominent in the phloem. Moreover, expression of an Escherichia coli-soluble pyrophosphatase in the phloem (pCoYMV::pyrophosphatase) of avp1-2 plants resulted in severe dwarf phenotype and abnormal leaf morphology. We conclude that the Proton-Pumping Pyrophosphatase AVP1 localized at the plasma membrane of the sieve element-companion cell complexes functions as a synthase, and that this activity is critical for the maintenance of pyrophosphate homeostasis required for phloem function.

Over-expression of the Arabidopsis proton-pyrophosphatase AVP1 enhances transplant survival, root mass, and fruit development under limiting phosphorus conditions.

Phosphorus (P), an element required for plant growth, fruit set, fruit development, and fruit ripening, can be deficient or unavailable in agricultural soils. Previously, it was shown that over-expression of a proton-pyrophosphatase gene AVP1/AVP1D (AVP1DOX) in Arabidopsis, rice, and tomato resulted in the enhancement of root branching and overall mass with the result of increased mineral P acquisition. However, although AVP1 over-expression also increased shoot biomass in Arabidopsis, this effect was not observed in tomato under phosphate-sufficient conditions. AVP1DOX tomato plants exhibited increased rootward auxin transport and root acidification compared with control plants. AVP1DOX tomato plants were analysed in detail under limiting P conditions in greenhouse and field trials. AVP1DOX plants produced 25% (P=0.001) more marketable ripened fruit per plant under P-deficient conditions compared with the controls. Further, under low phosphate conditions, AVP1DOX plants displayed increased phosphate transport from leaf (source) to fruit (sink) compared to controls. AVP1DOX plants also showed an 11% increase in transplant survival (P<0.01) in both greenhouse and field trials compared with the control plants. These results suggest that selection of tomato cultivars for increased proton pyrophosphatase gene expression could be useful when selecting for cultivars to be grown on marginal soils.

Enhanced proton translocating pyrophosphatase activity improves nitrogen use efficiency in Romaine lettuce.

Plant nitrate (NO3(-)) acquisition depends on the combined activities of root high- and low-affinity NO3(-) transporters and the proton gradient generated by the plasma membrane H(+)-ATPase. These processes are coordinated with photosynthesis and the carbon status of the plant. Here, we present the characterization of romaine lettuce (Lactuca sativa 'Conquistador') plants engineered to overexpress an intragenic gain-of-function allele of the type I proton translocating pyrophosphatase (H(+)-PPase) of Arabidopsis (Arabidopsis thaliana). The proton-pumping and inorganic pyrophosphate hydrolytic activities of these plants are augmented compared with control plants. Immunohistochemical data show a conspicuous increase in H(+)-PPase protein abundance at the vasculature of the transgenic plants. Transgenic plants displayed an enhanced rhizosphere acidification capacity consistent with the augmented plasma membrane H(+)-ATPase proton transport values, and ATP hydrolytic capacities evaluated in vitro. These transgenic lines outperform control plants when challenged with NO3(-) limitations in laboratory, greenhouse, and field scenarios. Furthermore, we report the characterization of a lettuce LsNRT2.1 gene that is constitutive up-regulated in the transgenic plants. Of note, the expression of the LsNRT2.1 gene in control plants is regulated by NO3(-) and sugars. Enhanced accumulation of (15)N-labeled fertilizer by transgenic lettuce compared with control plants was observed in greenhouse experiments. A negative correlation between the level of root soluble sugars and biomass is consistent with the strong root growth that characterizes these transgenic plants.

Arabidopsis sodium dependent and independent phenotypes triggered by H⁺-PPase up-regulation are SOS1 dependent.

Coordinate regulation of transporters at both the plasma membrane and vacuole contribute to plant cell's ability to adapt to a changing environment and play a key role in the maintenance of the chemiosmotic circuits required for cellular growth. The plasma membrane (PM) Na⁺/H⁺ antiporter (SOS1) is involved in salt tolerance, presumably in sodium extrusion; the vacuolar type I H⁺-PPase AVP1 is involved in vacuolar sodium sequestration, but its overexpression has also been shown to alter the abundance and activity of the PM H⁺-ATPase. Here we investigate the relationship between these transporters utilizing loss-of-function mutants of SOS1 (sos1) and increased expression of AVP1 (AVP1OX). Heightened expression of AVP1 enhances pyrophosphate-dependent proton pump activity, salt tolerance, ion vacuolar sequestration, K⁺ uptake capacity, root hair development, osmotic responses, and PM ATPase hydrolytic and proton pumping activities. In sos1 lines overexpressing AVP1, these phenotypes are negatively affected demonstrating that sos1 is epistatic to AVP1. Enhanced AVP1 protein levels require SOS1 and this regulation appears to be post-translational.

A transgenic approach to enhance phosphorus use efficiency in crops as part of a comprehensive strategy for sustainable agriculture.

Concerns about phosphorus (P) sustainability in agriculture arise not only from the potential of P scarcity but also from the known effects of agricultural P use beyond the field, i.e., eutrophication leading to dead zones in lakes, rivers and coastal oceans due to runoffs from fertilized fields. Plants possess a large number of adaptive responses to P(i) (orthophosphate) limitation that provide potential raw materials to enhance P(i) scavenging abilities of crop plants. Understanding and engineering these adaptive responses to increase the efficiency of crop capture of natural and fertilizer P(i) in soils is one way to optimize P(i) use efficiency (PUE) and, together with other approaches, help to meet the P sustainability challenge in agriculture. Research on the molecular and physiological basis of P(i) uptake is facilitating the generation of plants with enhanced P(i) use efficiency by genetic engineering. Here we describe work done in this direction with emphasis on the up-regulation of plant proton-translocating pyrophosphatases (H(+)-PPases).

Enhanced phosphorus nutrition in monocots and dicots over-expressing a phosphorus-responsive type I H+-pyrophosphatase.

Plants challenged by limited phosphorus undergo dramatic morphological and architectural changes in their root systems in order to increase their absorptive surface area. In this paper, it is shown that phosphorus deficiency results in increased expression of the type I H+-pyrophosphatase AVP1 (AVP, Arabidopsis vacuolar pyrophosphatase), subsequent increased P-type adenosine triphosphatase (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. However, AVP1 over-expression and the resulting rhizosphere acidification do not result in increased sensitivity to AlPO4, apparently because of the enhancement of potassium uptake and the release of organic acids. Thus, the over-expression of type I H+-pyrophosphatases 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.

Plant proton pumps.

Chemiosmotic circuits of plant cells are driven by proton (H(+)) gradients that mediate secondary active transport of compounds across plasma and endosomal membranes. Furthermore, regulation of endosomal acidification is critical for endocytic and secretory pathways. For plants to react to their constantly changing environments and at the same time maintain optimal metabolic conditions, the expression, activity and interplay of the pumps generating these H(+) gradients have to be tightly regulated. In this review, we will highlight results on the regulation, localization and physiological roles of these H(+)- pumps, namely the plasma membrane H(+)-ATPase, the vacuolar H(+)-ATPase and the vacuolar H(+)-PPase.

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

Engineering drought -resistant crop plants is a critically important objective. Overexpression of the vacuolar H(+)-pyrophosphatase (H(+)-PPase) AVP1 in the model plant Arabidopsis thaliana results in enhanced performance under soil water deficits. Recent work demonstrates that AVP1 plays an important role in root development through the facilitation of auxin fluxes. With the objective of improving crop performance, we expressed AVP1 in a commercial cultivar of tomato. This approach resulted in (i) greater pyrophosphate-driven cation transport into root vacuolar fractions, (ii) increased root biomass, and (iii) enhanced recovery of plants from an episode of soil water deficit stress. More robust root systems allowed transgenic tomato plants to take up greater amounts of water during the imposed water deficit stress, resulting in a more favorable plant water status and less injury. This study documents a general strategy for improving drought resistance of crops.

Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump.

Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na+ ions into the vacuole through the action of tonoplast proton pumps (an H+ -ATPase in the case of yeast, and either a H+ -pyrophosphatase (H+ -PPase) or H+ -ATPase in the case of plants) and a Na+/H+ antiporter is one mechanism that confers salt tolerance to these organisms. The cloning and characterization of genes encoding these tonoplast transport proteins from crop plants may contribute to our understanding of how to enhance crop plant response to saline stress. We cloned wheat orthologs of the Arabidopsis genes AtNHX1 and AVP1 using the polymerase chain reaction and primers corresponding to conserved regions of the respective coding sequences, and a wheat cDNA library as template. The wheat NHX cDNA cloned by this approach was a variant of the previously reported TNHX1 gene. The vacuolar H+ -PPase pump we cloned (TVP1) is the first member of this gene family cloned from wheat; it is deduced translation product is homologous to proteins encoded by genes in barley, rice, and Arabidopsis. Function of TNHX1 as a cation/proton antiporter was demonstrated using the nhx1 yeast mutant. TNHX1 was capable of suppressing the hyg sensitivity of nhx1. Functional characterization of the wheat H+ -PPase TVP1 was demonstrated using the yeast ena1 (plasma membrane Na+ -efflux transporter) mutant. Expression of TVP1 in ena1 suppressed its Na+ hypersensitivity. Expression analysis of salt-stressed wheat plants showed substantial up-regulation of TNHX1 transcript levels as compared to control plants, while transcript accumulation for TVP1 was not greatly affected by exposure of plants to salt stress.