In situ sensing of water potential

#The present invention relates generally to biosensors and systems for determining water potential in a substance, for example for in situ sensing of water stress in plants, and for high-throughput phenotyping.

#Changing climatic conditions pose numerous threats to humanity but none as severe as food insecurity. World Food Situation, Food and Agriculture Organization of the United Nations, FAO Cereal Supply and Demand Brief (2015). The increased frequency and severity of drought and high temperature conditions makes it imperative to develop crop plants that are able to cope with these environmental challenges in a sustainable manner. Sinclair T. R., HANDBOOK OF CLIMATE CHANGE AND AGROECOSYSTEMS: IMPACTS, ADAPTATION, AND MITIGATION, eds. D. Hillel & C. Rosenzweig, Imperial College Press (2010). In order to develop plants with effective water use (EWU) as well as to understand the physiological and genetic basis of abiotic stress tolerance, the ability to screen the response of large plant populations to stress is needed. Relative to the rapid evolution of genotyping technologies, the development of phenotyping tools for complex traits has lagged, hampering the study of key stress-adaptive traits and leading to an extensive knowledge gap. Richards et al., “Breeding for Improved Water Productivity in Temperate Cereals: Phenotyping, Quantitative Trait Loci, Markers and the Selection Environment,” Functional Plant Biology 37:85-97 (2010). Leaf water potential (Ψleaf) represents the best single indicator of plant water status, because it integrates environmental conditions (e.g., water availability and evaporative demand) and plant responses (e.g., stomatal regulation). Nobel P. S., PHYSICOCHEMICAL AND ENVIRONMENTAL PLANT PHYSIOLOGY. 2nd ed., Academic Press (1999). To date, techniques for the measurement of Ψleaf have been too cumbersome and expensive to be exploited in high throughput phenotyping strategies.

#Breeding and Phenotyping Strategies for EWU. Traditional breeding approaches that focus on selecting for yield within drought-prone or variable environments suffer from strong genotype-by-environment interactions (GxE) that inhibit the selection of superior genotypes and the potential for identifying useful genetic markers. Richards R. A., “Physiological Traits Used in the Breeding of New Cultivars for Water-Scarce Environments,” Agricultural Water Management 1:197-211 (2006). This realization has motivated efforts to identify physiological traits that control EWU as well as methods to measure them in a high-throughput manner. While there exist important examples of success (e.g., based on axial xylem resistance in wheat and anthesis-silking interval in maize), the gains in yield have been modest and the generality of these traits as useful indicators of EWU across species and environments remains unclear. Richards R. A., “Physiological Traits Used in the Breeding of New Cultivars for Water-Scarce Environments,” Agricultural Water Management 1:197-211 (2006). More recently, measurements of root structure have been pursued, but tend to require significant manual intervention for field-grown plants and have yet to provide significant improvements in EWU. Van Oosterom et al., “Hybrid Variation for Root System Efficiency in Maize: Potential Links to Drought Adaptation,” Functional Plant Biology 43:502-511 (2016); Tuberosa R., “Phenotyping for Drought Tolerance of Crops in the Genomics Era,” Frontiers in Physiology 3:347 (2012). Investigators have considered traits more directly related to water relations, for example, limited transpiration, but predict that strong GxE effects would need to be accounted for. Messina et al., “Limited-Transpiration Trait May Increase Maize Drought Tolerance in the US Corn Belt,” Agronomy Journal 107:1978-1986 (2015). Farquhar's and Richards' phenotyping methods based on using carbon isotope discrimination as a surrogate for water use efficiency stands out for its relative generality and ease of implementation. Farquhar et al., “Isotopic Composition of Plant Carbon Correlates with Water-Use Efficiency of Wheat Genotypes,” Functional Plant Biology 11:539-552 (1984). Nonetheless, isotope discrimination remains impractical for implementation on large mapping populations and can suffer from significant GxE effects. Richards R. A., “Physiological Traits Used in the Breeding of New Cultivars for Water-Scarce Environments,” Agricultural Water Management 1:197-211 (2006); Cordon et al., “Carbon Isotope Discrimination is Positively Correlated with Grain Yield and Dry Matter Production in Field-Grown Wheat,” Crop Science 27:996-1001 (1987). Continued progress in the development of water efficient crops for a changing climate will depend on new methods to define physiological traits associated with EWU.

#FIGS. 1A-1D depict aquaDust-enabled high-throughput phenotyping for Effective Water Use (EWU). FIG. 1A is a diagram showing aquaDust (shaded in dark gray) deployed in mesophyll such that leaf water potential (Ψleaf) can be sensed via calibrated reflectivity or fluorescence. FIG. 1B shows porous silicon-based aquaDust with strong, tunable dependence of reflectance on water potential (vapor activity). FIG. 1C shows the proposed mechanism of hydrogel-based fluorescent aquaDust based on the relationship of FRET with water potential via hydration state of gel. FIG. 1D measures EWU phenotypes based on multi-parameter envirotyping which can be integrated using crop growth models for genetic mapping of EWU to identify causal genes (μTensiometer is a Cornell technology). Pagay et al., “A Microtensiometer Capable of Measuring Water Potentials Below −10 MPa,” Lab on a Chip 14:2806-2817 (2014), which is hereby incorporated by reference in its entirety. The leaf clamp provides direct, spectroscopic measurements of aquaDust and chlorophyll fluorescence. UAV-mounted imaging provides proximal measurements.

#FIG. 2 illustrates intrinsic Water Use Efficiency (WUE) which is the rate of carbon fixed per unit mole of water transpired. Assimilation rate of carbon (A) is proportional to the difference of intercellular CO2 pressure (pi) and atmospheric CO2 pressure (pa) and the amount of water transpired (E) is driven by the difference between relative humidity within the leaf (ei) and outside the leaf (ea). WUA=A/E. A is represented by the arrow going in to the leaf. E is represented by the arrow going out of the leaf.

#FIGS. 3A-3B show false colored fluorescence images for bead size: 100 nm (FIG. 3A) and 10 nm (FIG. 3B), 3 cm away from the spot of injection in a maize leaf.

#One aspect of the present invention relates to a method for determining water potential in a substance. The method comprises contacting a substance with a biosensor, where the biosensor comprises a material capable of giving a detectable response to changes in local water potential and detecting the detectable response thereby determining water potential in the sub stance.

#Water potential (Ψ) is a form of chemical potential of water. It is the most widely measured plant water status parameter and is a useful indicator to predict plant and fruit growth, yield, and fruit composition and quality. One advantage of using water potential to quantify plant water status is that at equilibrium, both liquid and vapor (gas) phases have identical values of water potential.

#Plant water relations are governed by chemical (e.g., water) potential and its gradients, not by water content. There have been many studies of the effects of water stress on plant productivity and product quality though these have not always led to predictive tools. The main problem is that in-water status is very dynamic, both daily and seasonally, as plant water stress responds to both soil moisture and atmospheric evaporative demand. Ideal measures must be direct measurement of chemical potential, continuous to capture the dynamics, and inexpensive enough to allow spatial resolution.