Performing Department
Botany and Plant Sciences
Non Technical Summary
Plant nanobiotechnology is an emerging field of science that aims to utilize nanomaterials for studying plant organelles, tissues, and whole organisms and provide them with augmented functions. The Giraldo Lab works at the interface of plant physiology and nanotechnology to create new tools for studying how plants function and enhance plant productivity. We are initially focusing on three main programs to accomplish this overarching goal: 1) targeted and controlled delivery of biomolecules with nanoparticles in plants; 2) photonic nanomaterials for studying and enhancing the light and carbon reactions of photosynthesis; and 3) nanosensors for plant sugars and signaling molecules. Effective nanoengineering of plant function requires targeted and controlled delivery of nanoparticles with their cargoes to organelles and tissues of interest. Our research has highlighted that the charge and size of nanoparticles determines their uptake through isolated chloroplast lipid bilayers. To date the effect of nanoparticle structure and composition on the absorption, transport and distribution of nanomaterials in plants is poorly understood. My lab is exploring the mechanisms of nanoparticle targeted and controlled release of small biomolecules to specific plant tissues. We are interested in small biomolecules such as synthetic elicitors, drug-like compounds that induce natural immune responses in plants contributing to efficient regulation of plant protection against pathogens. The controlled induction of plant defense response by nanoparticles carrying synthetic elicitors can increase plant productivity by protecting crops from diseases without the need to be directly toxic to pathogenic organisms or hazardous to humans. Nanomaterials targeted to the chloroplasts could also enable the manipulation of other plant functions such as photosynthesis. Recently, we demonstrated that a plant nanobiotechnology approach enhances electron transport rates in extracted spinach chloroplasts and Arabidopsis leaves as a result of the spontaneous penetration of carbon and ceria based nanoparticles. We are now exploring the mechanisms of delivery of these nanoparticles to chloroplasts in vivo in crop plants such as tomato and cucumber, how they enhance the light reactions of photosynthesis, and their potential impact on plant carbon assimilation. Ultimately, we seek to integrate our plant nanobiotechnology approach with precision agriculture to enhance crop yields. We aim at regulating and monitoring individual plants needs for light, water, nutrients and defense elicitors using nanosensors. My work has demonstrated that nanosensors can detect changes in analyte concentration in living plants and report them to a commercially available near infrared camera. We expect that developing and implementing this plant nanobiotechnology approach will transform the field of plant physiology research and enable new nanotechnologies for crop yield enhancement in precision agriculture. These new technologies will contribute towards the Agricultural Experimental Station goals of developing knowledge that ensures continuous supply of food to a growing population while reducing the adverse effects on the environment and the consumer.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
1) Targeted and controlled delivery of biomolecules by nanoparticles in plants: Agricultural practices rely on spraying and irrigation technologies which end up inefficiently distributing chemical compounds such as fertilizers and pesticides in plants. Nanoparticles have been identified as effective delivery platforms for biomolecules in mammalian systems and more recently in plants. Understanding how nanoparticles can target specific plant tissues may lead to the efficient delivery and controlled release of biomolecules such as defense elicitors while avoiding plant products for human consumption. Developing mechanisms of endogenous or exogenous control of the timing of the defense response with nanomaterials will enable high precision tools to engineer the plant defense system. Silica nanoparticle's honeycomb-like porous structure, large surface areas, and biocompatibility make them particularly promising candidates for biochemical delivery in plants. However, it is not well understood how silica nanoparticles are transported through the intricate matrix of cellulose and pectin in plant cell walls, their interactions with subcellular structures and vascular system. Whereas targeted delivery of carbon nanotubes has been demonstrated in mice, to our knowledge it has not being reported in whole plants. Controlled release of small biomolecules by nanoparticles has been shown in animal cells and only recently in planta. We envision nanoparticle systems that control plant physiological responses in specific tissue locations, either by exogenous or endogenous stimuli, such as magnetic fields, enzyme activity, redox gradients, or specific analytes. A key question towards our goal of enhancing plant function with nanotechnology is how nanoparticle size, charge, and coating affect their transport within leaf tissues, and their interactions along the way with cell membranes and walls, mesophyll cells, and xylem vessels. Giraldo has shown that nanoparticles can be infiltrated through the leaf lamina of Arabidopsis plants and reach organelles like chloroplasts but the mechanisms of nanoparticle delivery and distribution within leaf tissues remain unknown.Our main objectives of this aim are: A) Understand the mechanisms of adsorption, transport, and distribution of nanoparticles varying in size, aspect ratios, charge, chemical and coating within leaf tissues. B) Functionalize nanoparticles for targeted delivery to specific tissues of biomolecules such as defense elicitors synthesized by our collaborator Prof. Thomas Eulgem from UCR. C) Enable controlled release of defense compounds by coating nanoparticles with magnetic nanomaterials or redox responsive gatekeepers.2) Nanomaterials for studying and enhancing the light and carbon reactions of plant photosynthesis: The mechanisms of enhancement of the light reactions of photosynthesis by nanoparticles are not well understood. The increase in chloroplast electron transport rates (ETR) by semiconducting single walled carbon nanotubes (SWCNT) is consistent with a broadening of the chloroplast photoabsorption spectrum. Semiconducting SWCNT convert photons into electron hole pairs (excitons) that could be transferred to photosystems or chlorophyll pigments. The semiconducting SWCNT capture light throughout the entire solar energy spectrum, including the green and near infrared (NIR) where chloroplasts have a weak absorbance. An alternative explanation is that the high electrical conductivity of SWCNT is responsible for boosting ETR between photosystems II and I. Metallic SWCNT do not generate excitons but may participate in slightly increasing chloroplast ETR. Whereas these studies were performed in extracted chloroplasts, the effect of semiconducting and metallic SWCNT on leaf ETR remains to be determined. Similarly, nanoceria particles were shown to significantly reduce levels of ROS in extracted chloroplasts but it is not known if and how nanoceria can affect the light reactions of leaves in living plants. Nanoceria are well positioned to complement the natural ROS scavenging mechanisms of plants by catalytically reducing ROS levels under stress conditions. It is also intriguing to think that SWCNT enhanced ETR or nanoceria ROS scavenging in leaves can result in higher plant assimilation rates. While SWCNT have been shown to colocalize with chloroplast photosynthetic pigments in leaves, it remains to be determined whether nanoceria can reach these sites of ROS generation in the chloroplast thylakoid membranes in planta. The creation of plants with carbon nanotube antennae and chemoprotective nanoceria particles would be transforming for agriculture, giving rise to nanoengineered crop plants with higher productivity.This aim focuses on understanding the mechanisms of nanomaterial enhancement of plant photosynthesis to create plants with higher productivity. To achieve this aim we outlined the following objectives: A) Investigate the mechanisms of semiconducting and metallic SWCNT enhancement of ETR. B) Determine the effect of ROS scavenging nanoceria particles on the light reactions of photosynthesis in leaves. C) Test the impact of SWCNT and nanoceria on leaf carbon assimilation and plant productivity.3) Nanosensors for plant sugars and free radicals: Precision agriculture aims to enhance plant productivity by closely monitoring and responding to individual plants needs for light, water, nutrients, and pathogen defense. Nanoparticles are promising candidates to act as sensors for reporting plant productivity, water status, and pathogen infection in real-time. Silver nanoparticles enhance the unique Raman signal of biomolecules by orders of magnitude, thus overcoming major limitations of optical sensors in complex living systems such as low specificity, high scattering background, and low signal to noise ratios. However, to date the application of silver nanoparticles as nanosensors in living plants has not been explored due to their instability in living organisms that leads to toxic effects. The Yin lab at the Chemistry Department of UCR has made a new nanoparticle alloy of silver and gold (SGNP) that is highly stable but maintains its capabilities of high Raman enhancement. SGNP excitation and emission in the NIR can generate an optical signal at wavelengths in which plant tissues are transparent. This NIR signal from embedded nanosensors in plant tissues can in turn be recorded by a confocal Raman spectrometer. Similarly, SWCNT have been demonstrated to detect single particles in plants in real-time but to date the microscopy systems able to detect the SWCNT fluorescence cannot spatially resolve their NIR signal at different depths in living tissues. To address this problem, we are currently designing and building the first confocal microscope able to capture the NIR signal from SWCNT in living organisms in collaboration with Dr. Sebastian Kruss from Gottingen University.My lab plans to utilize existent Raman spectroscopy equipment at UCR and build a new confocal microscopy system for 3D mapping nanosensors for plant sugars and free radicals. These cutting edge imaging technologies could translate into future monitoring applications for precision farming. We envision NIR cameras able to record the nanosensor signals in response to plant sugar production and stress. Thus improving real-time feedback of plant status for regulating soil nutrient content, pathogen control, water irrigation, among other factors affecting plant growth. With this goal on mind we plan to: A) Map the response of Raman silver-gold nanosensors to sugars in living plants. B) Build the first confocal microscope for imaging the NIR signal from carbon nanotubes in living tissues. C) Image in 3D the SWCNT based sensors for free radicals such as nitric oxide and hydrogen peroxide in plant tissues.
Project Methods
1) Targeted and controlled delivery of biomolecules by nanoparticles in plants: We will adapt tools from the pharmacokinetics field to understand how nanoparticles distribute in plants. We will manipulate the nanoparticle size, surface chemistry, and aspect ratios, and quantify parameters such as nanoparticle concentration over time, volume distribution, and transport rates in plant tissues of Arabidopsis, tomato, and cucumber plants. Nanoparticles labeled with fluorescent dyes will be imaged in the UCR Keen Hall Microscopy Core facility with Leica SP5 and SP2 confocals. We will produce the first datasets of nanoparticle rejection curves, rates of elimination, and bioavailability at cellular and tissue levels.By regulating nanoparticle size, charge, aspect ratios, and labeling the nanoparticle surface with peptides, we will constrain their delivery to target leaf tissues. We expect nanoparticles larger than 20 nm to be confined to the leaf extracellular air spaces whereas smaller nanoparticles would be transported to mesophyll and epidermal cells, stomata, and the leaf vascular system. We will explore the use of pathogen-derived ligands to target nanoparticles to plant cells expressing immune receptors. Plant pattern recognition receptors (PRRs) are a class of membrane-resident receptor-like protein kinases with an extracellular ligand binding domain that are upregulated under pathogen attack. Arabidopsis immune receptor FLS2 is a PRR which recognizes a fragment of the bacterial protein flagellin termed flg22. FLS2 is preferentially expressed in plant tissues particularly strongly exposed to pathogens, such as stomata and leaf vasculature. We will direct nanoparticles carrying synthetic elicitors to stomata guard cells and minor veins exposed to pathogen attack by coating them with flg22. We will demonstrate that nanoparticles reach their expected target with confocal imaging, using fluorophores to label plant tissues and nanoparticles. To examine the efficiency of our strategy we will perform standard pathogen defense assays.We plan to use nanoparticles that act as controlled gatekeepers of small molecules such as plant defense elicitors in response to endogenous signals like redox gradients or exogenous signals such as magnetic fields. For creating redox-sensitive nanoparticle systems, the silica nanoparticle amphiphilic copolymer corona will contain disulfide links within the hydrophobic backbone that are prone to cleavage by gluthatione. For exogenous control of defense compounds, we will use silica shell nanocarriers consisting of a magnetic core (Fe3O4) coated with a thermal sensitive polymer. An alternate magnetic field can induce an increase in temperature in these magnetic nanocarriers releasing their cargo.2) Nanomaterials for studying and enhancing the light and carbon reactions of plant photosynthesis: Leaves of Arabidopsis, tomato, and cucumber plants will be infiltrated with a mix of SWCNT, metallic m-SWCNT, and semiconducting s-SWCNT. These SWCNT will be suspended in deionized water with single stranded DNA. DNA-SWCNT will then be dissolved in MES or TES buffers and delivered to Arabidopsis, tomato, and cucumber leaf mesophyll cells by infiltration through the stomata. To determine if SWCNT can reach the chloroplasts in planta, the SWCNT will be labeled with fluorescent dyes and leaf punches imaged with a Leica SP5 confocal. A 3D rendering of the confocal images will be constructed and analyzed in Fiji. The ETR of leaves infiltrated with SWCNT and buffer (controls) will be measured with a fluorometer (GFS-3000 FL, Walz) from saturating light levels to dark. The s-SWCNT with peaks of absorption in the green and the NIR will be used to determine if these wavelengths drive the enhancement of ETR by SWCNT. Oxygen evolution will be quantified in leaves with semiconducting and metallic SWCNT with a needle type oxygen sensor (Pyroscience). By blocking the electron transport between PSII and PSI via plastoquinone with (3-(3,4-dichlorophenyl)-1,1-dimethylurea) DCMU, we will test if m-SWCNT can act as a bridge between these photosystems. Together these experiments will address fundamental questions of how SWCNT affect light energy capture by the photosystems, the source of increased electron flow, and the effect of SWCNT as conductors of electrons in the thylakoid membranes.My lab is also determining the effect of ROS scavenging by nanoceria particles on the leaf light reactions of photosynthesis. Nanoceria particles will be coated with biocompatible polymers of negative and positive zeta potentials to determine if surface charge affects their localization in chloroplasts in planta as it was observed in extracted chloroplasts. Infiltration of nanoceria particles in leaf tissues and confocal imaging will be performed as explained above. The ETR of leaves infiltrated with nanoceria particles and buffer (controls) will be measured with a GFS-3000 FL from bright light (2000 μmol m-2 s-1) to dark. The quantum yield of PSII will be measured in dark adapted leaves to assess the health of these photosystems. The chlorophyll content will be monitored using a MC-100 chlorophyll index meter (Apogee). Leaf production of ROS such as hydrogen peroxide, singlet oxygen, and superoxide will be imaged in vivo with a Leica SP5 confocal microscope using fluorescent probes. Levels of antioxidants such as ascorbic acid (AsA) and dehydroascorbate (DHA) will be measured by spectrophotometry.Light curves of carbon assimilation will be measured with a GFS-3000 FL in leaves infiltrated with mixed SWCNT, s-SWCNT, and m-SWCNT under different light sources of white, green, and NIR. Nanoceria particles are expected to protect the plant photosynthetic machinery from ROS under high photon fluency, thus it is likely that light curves of carbon assimilation would indicate that leaves with nanoceria sustain higher levels of assimilation. In addition, the maximum rate of carboxylation of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in leaves with s-SWCNT, m-SWCNT, and nanoceria particles will be calculated from photosynthetic CO2 response curves to analyze the role of the nanoparticles in maintaining the activity of Rubisco.3) Nanosensors for plant sugars and signaling molecules: Silver gold nanoparticle alloys will be synthesized by the Yadong Yin lab. The SGNP corona will be functionalized with mercaptophenylboronic acids having a high affinity to glucose and sucrose. Phenylboronic acid functional groups could link to a variety of plant sugars, including some sugar residues in polymers in the cell wall, instead of just to glucose and sucrose. We will identify characteristic Raman peaks for these sugars with the SGNP to differentiate them in plant tissues. Ultra-fast hyperspectral imaging with nanoscale resolution of SGNP sensors in leaf tissues will be performed with SWIFTTM and tip enhanced confocal Raman spectroscopy. The nanoscale resolution will enable the mapping of sugars at a scale that separates the Raman signal of cytosol and organelle sugars from potential cell wall sugar residues. Excitation of nanosensors inside plants with a 785 nm laser will be off resonance of photosynthetic pigments.SWCNT will be functionalized to detect free radicals such as nitric oxide and hydrogen peroxide via a rapid coating exchange protocol we developed. We will infiltrate the SWCNT through the leaf lamina. For imaging SWCNT nanosensors for free radicals, in collaboration with Sebastian Kruss, we will mount and couple a Xenics NIR camera to his BX63 Olympus microscope equipped with a 561 nm laser and Olympus disk spinning unit. We will image the NIR fluorescence of SWCNT in response to nitric oxide and hydrogen peroxide added exogenously to the leaf lamina. The SWCNT signal will be colocalized with chloroplast autofluorescence, cell membranes and walls labeled with fluorescent dyes.