CuO and ZnO Nanoparticles Modify Interkingdom Cell Signaling

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Review

CuO and ZnO Nanoparticles Modify Interkingdom Cell Signaling Processes Relevant to Crop Production: A Review. Anne Anderson, Joan E. McLean, Astrid Rosa Jacobson, and David Britt J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 08 May 2017 Downloaded from http://pubs.acs.org on May 11, 2017

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Journal of Agricultural and Food Chemistry

CuO and ZnO Nanoparticles Modify Interkingdom Cell Signaling Processes Relevant to Crop Production: A Review *

Anne J Anderson1, Joan E McLean2, Astrid R Jacobson3, David W Britt4.

1 Department of Biology, Utah State University, Logan, Utah 84322- 5305 2 Department of Civil and Environmental Engineering, Utah Water Research Laboratory, Utah State University, Logan, Utah 84322 -8200 USA 3 Department of Plants, Soils and Climate, Utah State University, Logan, Utah 84322 USA 4 Department of Bioengineering, Utah State University, Logan, Utah 84322-4105 USA *Corresponding author [email protected]

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ABSTRACT: As the world population increases, strategies for sustainable agriculture

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are needed to fulfill the global need for plants for food and other commercial products.

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Nanoparticle formulations are likely to be part of the developing strategies. CuO and

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ZnO nanoparticles (NPs) offer potential as fertilizers, as they provide bioavailable

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essential metals, and as pesticides, because of dose-dependent toxicity. Effects of

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these metal oxide NPs on rhizosphere functions are the focus of this review. These NPs

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at doses of 10 mg metal/kg and above change production of key metabolites involved in

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plant protection in a root-associated microbe, Pseudomonas chlororaphis O6. Altered

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synthesis occurs in the microbe for phenazines, which function in plant resistance to

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pathogens, the pyoverdine-like siderophore that enhance Fe bioavailability in the

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rhizosphere and indole-3-acetic acid affecting plant growth. In wheat seedlings,

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reprograming of root morphology involves increases in root hair proliferation (CuO NPs)

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and lateral root formation (ZnO NPs). Systemic changes in wheat shoot gene

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expression point to altered regulation for metal stress resilience as well as the potential

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for enhanced survival under stress commonly encountered in the field. These

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responses to the NPs cross kingdoms involving the bacteria, fungi and plants in the

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rhizosphere. Our challenge is to learn how to understand the value of these potential

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changes and successfully formulate the NPs for optimal activity in the rhizosphere of

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crop plants. These formulations may be integrated into developing practices to ensure

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sustainability of crop production.

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KEY WORDS: root microbiome, metabolites, acyl homoserine lactones, siderophore,

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phenazine, Pseudomonas chlororaphis O6, indole-3-acetic acid, drought stress

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1. INTRODUCTION

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1.1.

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This review focuses on the impact of nanoproducts on key processes in the plant’s

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rhizosphere affecting plant health. The effects are observed both on the plant, and a

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common type of bacterium that colonizes the root surface. Basic laboratory studies at

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the cellular, biochemical and transcriptome levels show that CuO and ZnO NPs have an

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impact on metabolites acting as signals in the communication between the plant and the

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bacterial cells for functions in the rhizosphere. The work illustrates the need in the

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development of nanoproducts for agriculture to consider not only plant responses to

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NPs, but, also the essential role played by the plant’s microbiome in plant productivity.

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1.2.

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The overview of the review.

Potential uses of metal oxide NPs in agriculture. Nanosized materials will be engineered and formulated for agricultural use,

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although currently commercialization is in its infancy. Yield and quality of crops are

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being challenged by the growing human population, shrinking prime agricultural land

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and irrigation water, and weather instability. Additionally, there are problems associated

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with accumulation and run off from applications of the conventional commercial

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pesticides used in agriculture, industry and domestic settings. Many of the filed patents

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feature modification to nanosize particles of materials with proven efficacy as bulk

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products as fertilizers or pesticides. Nanomaterials also offer potential as delivery

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vehicles and as sensors for key processes in crop production.1-6 Entry of NPs into

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agricultural soils could also occur from purposely- applied sludges, or by accident, such

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as agricultural use of polluted water.7,8 A recent review indicated that sludge contained

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“vast stores of metal-containing nanoparticles”.8 Additionally, trophic transfer of NPs



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through natural food chains demonstrates that their applications to soils and plant

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extends exposure to multiple organisms from different kingdoms.9-11 As with

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applications of all chemicals in agriculture, a balance must be obtained between positive

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and negative consequences for the nanoagricultural formulations. Aspects of potential

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risks of the application of nanoproducts are extensively reviewed.12-16

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NPs containing essential metals, such as Fe, Mg, Zn, Cu and Mn, are proposed

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as fertilizers at low doses and as pesticides at higher doses 17-20 because these metals

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are vital for cellular function yet toxic above a threshold.21,22 Fertilization is important for

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soils where the bioavailability of essential metals is limited. Restricted availability could

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be due to lack of metal-containing minerals or the removal of the metal resources

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through continuous cropping of the soil. Also the metals could be present but held in the

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soil in nonbioavailable forms as minerals or with organic matter .23 Worldwide there are

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many soils producing plants deficient in Zn, leading to low Zn nutrition when they are

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used as foods.24-26 Zn deficiency is present in 30 % of the world’s population and

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impairs growth, reproduction, nervous system development and the immune

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system.25.26 Cu-deficient soils occur less frequently worldwide but are present 27in part

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due to low bioavailability because of mineral complexation23 and the strong association

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of Cu with organic matter.28,29 Symptoms of Cu stress occur on plants grown on the

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deficient soils such as wither tip of cereals.27

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The potential use as fertilizers for metal oxide NPs stems from increased metal

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accumulation in plants grown with the NPs in different matrices, including field soils 30-33

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(Fig. 1). Metal accumulations due to growth with ZnO and CuO NPs, uptake is due to

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both the plant responds to the NPs and the metal released by dissolution in the 4 ACS Paragon Plus Environment

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rhizosphere or in planta. Uptake of intact NPs into plants is observed, but with high

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variability between plant species. For instance, no ZnO NPs are detected in cells of

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soybean roots,34 whereas aggregates of ZnO NPs are detected in maize xylem.35 CuO

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NPs applications to maize roots result in transport of the NPs up into the leaves and

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then redistribution back to the roots.36 Translocation of NPs into shoots also occurs in

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rice grown with CuO NPs, although the Casparian strip acts as a physical barrier.32

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Although magnetite NPs are not transported into wheat shoots,37 uptake and transport

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of iron oxide NPs occurs in pumpkin.38 Intracellular trafficking also is reported. ZnO NPs

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are internalized by root cells of a wetland plant39 and TiO2 NPs enter maize root cells.40

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The degree of bioaccumulation of metal from CuO NPs is influenced by other factors

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such as weathering of the NPs.41 These variabilities illustrate the complexity of

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predicting the efficacy of the NPs in agricultural settings that are discussed in more

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detail in this review.

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At higher doses than those suitable for plant fertilization, toxic effects of Zn and

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Cu on cellular functions raise the possibility of their use as pesticides where pathogenic

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fungi and bacteria could be targeted. For instance inhibited growth of plant pathogenic

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fungi and oomycetes is demonstrated with dose dependent effects (e.g. >100 mg

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metal/L).42-46 Early and current work assessing toxic effects of NPs on bacteria focus on

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their potential as an alternative control strategy for human pathogenic bacteria, a

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strategy needed because of the development of resistance to traditional antibiotics.47

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Initial responses in cell toxicity may involve binding to cell walls.48 Changes to

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membrane potentials 49 and disturbance of the redox balance within a cell contribute to

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toxicity. The ability of the Cu ion to exist in two redox states catalyzes formation of



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superoxide anion due to reduction of molecular oxygen. Also both Zn and Cu interact

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with -SH groups, such as those found in proteins and a key intracellular antioxidant,

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glutathione. Inactivation of enzyme activity and depletion of glutathione contribute to

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cellular stress from accumulated reactive oxygen species (ROS).50,51 Indeed in a review

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of the impact of metal oxide NPs on plants focusses on detoxification of ROS as an

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important survival mechanism.13

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The positive value of nanoparticles (NPs) in agriculture is tempered by the need

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to predict negative effects, such as phytotoxicity and deleterious effects on essential soil

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microbe functions.12,16 Understanding at the molecular level, the basics involved in

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variability between responses with different NPs and plants and soils plus their

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microbiomes (Fig. 2) will promote confident use of nanoagricultural products. However,

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currently such studies at these levels are limited.52

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This review addresses, in a model system, cellular, biochemical and

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transcriptome changes induced through the presence of CuO and ZnO NPs in the

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rhizosphere on both the plant and root-colonizing bacterial cells. The work described

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has been performed under defined laboratory conditions in shake and agar plate culture

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for the microbial studies, and in growth boxes with sand or with soils for the plant

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studies. We do not know whether the responses observed with sublethal levels of CuO

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and ZnO NPs occur in agricultural field native soils. Rather, we review the findings to

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promote awareness of potential changes in plant-microbe signaling that are set in place

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with exposure of roots and their associated microbial cells to the NPs.

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1.3. The root microbiome. This review highlights that plants under field

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conditions grow in association with microbes. This fact contrasts to the many plant-NP 6 ACS Paragon Plus Environment

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studies where the role of a microbiome is not considered. Certain microbial plant

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colonizers exert changes in the plant that parallel those observed with microbes termed

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probiotics, which are essential for human health.53 Outcomes for the plant from

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colonization with the probiotic-like microbes include enhanced growth, changes in

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morphology, antagonism of microbial pathogens, stimulation of defense mechanisms

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against environmental stress and improved nutrition. Biofortification of soils with

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probiotic-like microbes will be likely be an important aspect of the practices employed

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for sustainable agriculture.54-57 Currently there are several commercial formulations of

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microbes sold as biofertilizers or biocontrol agents to combat microbial diseases.55,56

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Thus, it is interesting that there is overlap in agricultural use of these beneficial

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microbes as fertilizers and pesticides, with proposed uses for NPs.2,6,17-20

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Microbial populations are greater in rhizosphere, the region around the root

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influenced by exudation of plant metabolites, than in the surrounding soil.58 These plant

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exudates provide nutrients to support microbial growth.59-62 However, the complex

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microbial community structure in soils is disturbed by NP amendments, with both

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increases and decreases for certain taxa occurring dependent on NP, plant and soil

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properties.63,64 The soil microbes involved in the nitrogen cycle are among those with

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documented sensitivity.65-67 Changes in function of a population, such as a loss in

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function of nitrogen-fixing rhizobium isolates, would in turn affect plant

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performance.30,65,68 Clearly any formulation for agricultural use should not have

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deleterious effects on the organisms involved in essential element cycling.16

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The framework for discussion of NPs use in agriculture in this review centers on studies with a pseudomonad able to colonize the roots of many plant species, a process 7 ACS Paragon Plus Environment


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that improves the plants growth and stress resilience.69 Pseudomonas chlororaphis

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isolate O6 (PcO6) (Fig. 3) was cultured from the roots of commercial dry land wheat at

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the end of the growing season in calcareous soil. Like other pseudomonads, it is

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adaptable to using many substrates for growth utilizing the sugars, organic acid and

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amino acids that are major metabolites in all root exudates.58-62,70 PcO6 is typical of the

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many microbes with biological control activity.71-73 For example, colonization of plant

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roots with PcO6 confers systemic protection against viral, bacterial, and fungal

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pathogens in the plant. Protection also arises from direct antagonism of microbial

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pathogen growth caused by production of antibiotics.74-77 PcO6 is lethal to root knot

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nematodes, with HCN production playing a major role.78 Killing of the larval stage of

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certain insect pests is conditioned by production of an insecticidal peptide toxin, Fit

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D.72,79 Root colonization by PcO6 also stimulates in the host plant drought tolerance and

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resilience to heavy metals.80,81 Thus, the consequence of contributing C and N in root

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exudates to support colonization of beneficial microbes is the provision of an umbrella of

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protection for the plant against the array of stresses experienced in a field environment.

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However, such generalist root colonizers would be among the rhizosphere organisms

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challenged by NPs when introduced into agricultural soils.

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2. MODIFICATION OF PcO6 METABOLISM BY NPs

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2.1 The metal oxide NPs. In the studies discussed in the following sections, the CuO

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and ZnO NPs were used “as-made” from the manufacturer.31,45,46,70, 82 Nanodimensions

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of the particles were confirmed by microscopy and their chemistry by elemental analysis


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with ICP-MS, XRD and ZANES analysis.82 Aggregation of the particles, shape

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modification and dissolution under different conditions is reported.46,82

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2.2. ZnO NPs: Phenazine and AHSL production. PcO6 is an example of the

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biological control agents that produce the phenazine class of antibiotics (Fig. 3). The

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phenazines from PcO6 are visualized as orange pigments secreted from the bacterial

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cell 74 (Fig. 3). Their synthesis occurs from the shikimic acid pathway through

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chorismate.84 Nutrients, such as the sugars that feed into the shikimic acid pathway in

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the bacterial cell, are part of the exudates from germinating seeds and plant roots.69

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Thus, the C and N in the plant exudates is repurposed by bacterial metabolism to

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generate the phenazines which in turn are beneficial to plant and rhizosphere health.

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Phenazines originally were examined in vitro for their direct antifungal activities,

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becoming associated with disease suppression.85 Mutation, so that the bacteria lack

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phenazine production, reduces the extent of growth inhibition for plant pathogenic fungi

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by the pseudomonad.85 Phenazines are isolated from native fields soils in which wheat

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is grown at levels sufficiently high to cause antagonism of pathogen growth.86 This

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finding indicates that phenazine production is not just a laboratory phenomenon.

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Phenazines are involved in additional processes that affect plant function.87,88

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Phenazines are elicitors that prime systemic induced resistance in plants. “Priming”

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means that the plant responds faster and to a greater extent when subsequently

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challenged by a stress such as drought or pathogen attack. 76,89-91 Primed defense is

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observed systemically at sites distant from the inducing microbe. For instance, shoot

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tissues are protected whereas the inducing microbes are located only on the root

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surface.76 This systemic induction of resistance augments direct antagonism of 9 ACS Paragon Plus Environment


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pathogen growth. These responses illustrate that the phenazines are inter-kingdom

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signaling chemicals, affecting the functioning of bacteria, the plant as well as fungi in the

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environment.

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Effective biocontrol agents display aggressive and sustainable colonization of

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plant roots. One of the traits needed for sustained colonization is attachment to the root

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surface followed by growth of the cells embedded within a gel-like matrix as a

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biofilm92,93 (Fig. 1). The pseudomonad biofilms formed on plant root surfaces protect

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against challenges of the CuO NPs and ROS that are lethal to planktonic cells.81,94

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Resilience of biofilm cells to antibiotics generated by other rhizosphere microbes is

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likely.95 Aspects of biofilm formation are covered in a companion paper in this journal

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issue by Britt et al. (2017). Thus, biofilm matrix, with its intertwining polymers of

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polysaccharides, nucleic acids and proteins, provides a protective environment for

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microbial cell survival.

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Phenazines play additional roles in biofilm function. For instance, phenazine

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production in P. chlororaphis 30-84 enhances the extrusion of extracellular DNA levels

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which becomes part of the biofilm matrix.96 Another role of phenazines in biofilms is to

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accept electrons and, consequently, aid redox balancing in bacterial cells under

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anaerobic conditions, which are likely to occur within layered biofilms in the

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rhizosphere.97 Biofilm formation in pseudomonads is promoted by an adequate iron

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supply.98,99 Phenazines may through acting as iron chelates promote the release of

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metals from minerals,100,101 and, thus, enhance microbial biofilm formation. Additionally,

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their redox activity will reduce ferric to ferrous ions, which can be transported into cells

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by mechanisms that differ between these charged Fe ions.98 10 ACS Paragon Plus Environment

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Phenazine synthesis in pseudomonads is regulated by the Gac/Rsm signal

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transduction pathway. In PcO6, synthesis is dependent on acyl homoserine lactones

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(AHSLs) (Fig. 3) produced by activation of the Gac/Rsm pathway.88 Synthesis of an

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array of AHSLs with different fatty acid chain lengths and of families with the basic

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phenazine structure applies to PcO683 and other beneficial pseudomonads.102 When

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the bacterial cells reach a sufficiently high density, the AHSLs reprogram the bacterial

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cell population to act in unison to initiate phenazine synthesis; an event termed quorum

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sensing.103 ZnO NPs affect this process. A decrease in phenazine production occurs as

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ZnO NPs are added to broth medium (Fig. 4). 83,104 These effects were seen at 2 mg

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soluble Zn/L from ZnO NPs.83 AHSLs also were reduced in levels in broths with the

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ZnO NP-treatments (Fig. 4).83 The lack of AHSLs correlated to lowered expression from

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the AHSL synthase gene, phzI.83 Inhibition of phenazine and AHSLs production by the

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ZnO NPs is duplicated in part by Zn ions but not with the same efficacy as from the

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NPs.83 We speculate that the NPs are highly effective as a source of Zn for the bacterial

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cell through metal oxide dissolution. Dissolution of the ZnO NPs could be increased by

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metabolites produced by the bacterium that are metal chelators, such as siderophores

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(see Section 2.2) or gluconic acid produced by the bacterial cells.81

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Reduction of both phenazines and AHSLs by exposure to ZnO NPs could

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change the effects of potential pathogens in the rhizosphere. The AHSLs, like the

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phenazines, trigger systemic defense mechanisms in the plant.105,106 Thus, two different

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metabolites used in signal communication between plant and the rhizosphere microbe

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are modified in synthesis by the ZnO NPs. Whether other antibiotics produced by

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PcO6, which are regulated independently of AHSLs within the Gac/RSm network, are



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modified by the ZnO NPs awaits study. This work with PcO6 also raises the question of

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whether NPs have a similar impact on the metabolism of other rhizosphere bacteria that

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produce antimicrobial agents.

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2.3. ZnO NPs and siderophore production. Coincident with the loss of phenazine

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production when PcO6 is exposed to ZnO NPs is an increase in a secreted, fluorescent

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pyoverdine-like siderophore.107 (Fig. 3 and Fig. 4). The major function of siderophores is

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binding Fe3+ ions that are then transported and unloaded into the bacterial cell to

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provide this essential metal.108,109 Expression from the large biosynthetic cluster of

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genes involved in pyoverdine synthesis in pseudomonads is highly regulated, with

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suppression occurring when Fe is adequate.96 Although these biochemical changes

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were demonstrated in broth cultures, ZnO NPs also had an impact on siderophore

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production in PcO6 cells colonizing the roots of bean growing in a solid matrix.110

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Increased pyoverdine levels were detected in bean rhizosphere solutions with the

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presence of ZnO NPs in the plant growth matrix.110

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The mechanism underpinning altered expression from the siderophore genes is

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not clear. Increased pyoverdine production in PcO6 is observed in a mutant in which the

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GacS environmental signaling protein of the Gac/Rsm controlling network is

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eliminated.111 This is one of the few traits identified as being negatively regulated by the

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Gac/Rsm network in isolate PcO6; currently most of the traits studied for this network,

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such as production of phenazine and AHSLs, are positively regulated.88 Thus, the ZnO

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NPs appear to induce a gacS-like phenotype to the cells. In a gacS mutant, the

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translational repression, caused by the binding of the protein RsmA for genes in the gac

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regulon, cannot be relieved.112 Studies with P. aeruginosa (Pa) show that the 12 ACS Paragon Plus Environment

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translation inhibitory protein, RsmA, is a strong repressor of pyoverdine synthesis i.e.,

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deletion of RsmA in Pa increases pyoverdine production.113 This response is attributed

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to indirect transcriptional control by RsmA of the alternative sigma factor, PvdS, that is

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required for expression of the pyoverdine biosynthetic genes. Transcriptional regulation

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of pvdS is correlated with levels of the intracellular cell signaling compound, c-di-GMP,

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that are governed by RsmA; a Pa rsmA mutant has higher levels of c-di-GMP.113 This

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control network would explain the responses of PcO6 but is puzzling in light of other

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studies in Escherichia coli where Zn ions are inhibitory to protein diguanylate cyclase

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that produces c-di-GMP,114 meaning that with Zn inhibition, levels of c-di GMP would be

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lowered. Whether Zn modifies c-di-GMP levels in pseudomonad cells is not known.

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The increase in pyoverdine-like siderophore production from rhizosphere bacteria

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triggered by ZnO NPs (Fig. 4) may be advantageous to the plant. Several potential

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outcomes exist. First, the chelation efficiency for Fe of the siderophore could limit

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growth of pathogenic rhizosphere microbes. This possibility is likely because pyoverdine

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has high binding capacity for Fe. Indeed siderophore production from fluorescent

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pseudomonads was among the first traits to be associated with suppression of disease

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caused by fungal pathogens in the field.115,116 Second, plants may access the Fe from

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the loaded pyoverdine-like siderophores117-119 for nutritional benefit. Third, the

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pyoverdine-like siderophores also are among the microbial metabolites that stimulate

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systemic plant resistance mechanisms.120-122 Thus, under laboratory conditions

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although the ZnO NPs reduce production of the interkingdom cell signaling metabolites,

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phenazines and AHSLs from PcO6, there is enhanced production of another

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interkingdom signaling compound, a pyoverdine-like siderophore. If these phenomena



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occur in native soils, there is potential for changes in plant defense against pathogen

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attack.

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2.4. CuO NPs and PcO6 metabolism. In contrast to the increase by ZnO NPs in

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siderophore production by PcO6, CuO NPs decrease the pyoverdine levels (Fig. 4).123

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Analysis of gene expression in the PcO6 cells finds lower transcript abundance from

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genes encoding the cytoplasmic transporter and proteins involved in periplasmic

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maturation of the pyoverdine precursor with CuO NPs.123 Thus, the chemistries of these

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two metal oxide NPs elicit different cellular responses in the microbe.

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Opposing regulation in PcO6 in response to the two NPs also is observed with

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the production of a plant growth regulator IAA (Fig. 4). In PcO6, IAA is synthesized from

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the amino acid, tryptophan, which is among the array of amino acids detected in root

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exudates at levels varying between plant types.59 IAA production in PcO6 was

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enhanced by CuO NPs but repressed by ZnO NPs.124The mechanisms causing these

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effects await elucidation. However, further complexity with the Gac/Rsm regulon is

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evident because IAA production from tryptophan also is boosted by deletion of GacS in

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PcO6.125 Thus, stimulation of IAA production by ZnO NPs would be expected if these

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NPs produced a gacS-like phenotype. We speculate that there must be a strong

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inhibitory effect of Zn at a site currently unidentified in the tryptophan-dependent

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synthesis of IAA.

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IAA production by PcO6 cells colonizing wheat roots affects root morphology

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when the growth matrix is supplemented with tryptophan.126 The wheat roots shorten

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and prolific and elongated root hairs are observed, as reported for roots exposed to

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authentic IAA or other IAA-producing microbes.127 IAA production by root-associated 14 ACS Paragon Plus Environment

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microbes has field significance because this trait is one prevalent in isolates displaying

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plant growth promotion and the ability to enhance metal phytoremediation.128,129

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These studies illustrate that the metabolism of the rhizosphere-colonist PcO6 can

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be tuned by Cu or Zn supplied through NPs (Fig. 4). The metabolites that change in

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level affect functions of plants and fungi that are involved in plant health.

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3. PLANT RESPONSES TO NPs

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3.1. NPs and their impact on plant roots. Plants closely control Cu and Zn

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homeostasis. The physical mechanisms to limit toxicity differ between the metals:

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excess Cu is held largely within plant cell walls, whereas Zn is contained within

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vacuoles.130,131 Studies with CuO NPs indicate that the capture of Cu by root cell walls is

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important to limit Cu flux into the shoots of the rice plant.32 Both CuO and ZnO NPs

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increase lignification in roots of several plant species through processes that involve

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altered peroxidase activities.132-133 These changes may be a generalized response to

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increased ROS in the plant tissue due to enhanced metal levels.13

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The NPs also manipulate the plasticity of root morphology with outcomes that

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differ between the ZnO and CuO NPs (Fig. 5). Both outcome however would increase

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root surface area potentially providing more support for colonization by beneficial

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microbes. When grown with CuO NPs, the roots of wheat seedlings are shortened in

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length (e.g., from 12-14 cm for controls to less than 4 cm with CuO NPs depending on

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dose) and there is prolific elongated root hair formation close to the root tip114 (Fig. 5).

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With sand as the growth matrix for 7 d-wheat seedlings, these morphological changes

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occur at doses of 10 mg Cu/kg sand.126 Shortening of root length in wheat is extreme in

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acidic soils (Fig. 5) and correlates with dissolution of CuO NPs, whereas in calcareous 15 ACS Paragon Plus Environment


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soil there is mitigation of toxicity.26 The changes in root hair patterning may not be

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universal for plants; for instance, Cu ion treatment of cowpea also causes root stunting

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but the root hairs become shortened.134

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The gross morphological changes observed in wheat tips grown with CuO NPs,

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resemble those from high IAA levels.26 We speculate that the dissolution of CuO NPs in

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the root tip zone allows Cu to accumulate to a level that affects the normal inverted

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fountain flow of IAA as it unloads from the stele. Root exposure to Cu ions is reported to

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inhibit a specific efflux pump in the plant cells at the base of the stele, causing IAA flux

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in the root tip cells to be modified.135

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The CuO NPs were more effective in induction of root hair proliferation than Cu

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ions, but this could be attributed to dilution of the ion concentration in the rhizosphere

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space below that of the gradient of ions being released from NP aggregated on the root

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surface.113 The phenomenon of CuO NP-enhanced root hair formation, could benefit

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plants in the field because root hairs are involved in nutrient and water uptake.136,137 It is

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possible that the entrapment of the CuO NPs onto the root hairs (Fig. 1), as revealed by

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microscopic imaging,114 is important to this phenomenon.

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3.2. Lateral root growth proliferation. Growth with ZnO NPs has no effect on the

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differentiation of epidermal cells into root hairs. However, lateral roots are induced when

340

wheat seedlings are grown in sand and calcareous soils (Fig. 5).31 Lateral root formation

341

is dependent on Zn in other plants138 and consequently the changes in wheat caused by

342

the ZnO NPs may depend on their dissolution in the rhizosphere or root tissues. The

343

molecular basis of stimulation of lateral root primordia is not fully understood, although

344

oscillations in IAA in the root tip are proposed to be involved and IAA signaling is 16 ACS Paragon Plus Environment

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345

required in the transforming cells.139-141 Clearly, any changes in IAA triggered by ZnO

346

NPs must differ from those caused by CuO NPs because the resulting root

347

morphologies are distinct (Fig. 5). Stimulated lateral root formation by formulations with

348

ZnO NPs could also be valuable under field conditions because they will increase

349

nutrient and water uptake. It is notable that lateral root hairs are especially involved in

350

uptake of Si that increases plant strength and resilience to pathogens.142

351

3.3. CuO and ZnO NPs and their impact on plant’s response to stress. At doses of

352

ZnO or CuO NPs that cause changes in root morphology (300 mg M/kg), there are

353

effects on transcript accumulations in the roots, as well as systemically in the shoots

354

(Anderson et al, unpublished). Transcript analysis in the roots show increased

355

expression of genes associated with resistance to metal stress incited by ion

356

exposure.81 These findings suggest that the plant perceives metal ions released from

357

the NPs. Colonization of the plant root with PcO6 reduces the extent of transcript

358

abundance, about 2-3 fold.81 We speculate that the lowered transcript levels could be

359

due a protective effect of the PcO6 biofilm (Fig. 1) on the root in which NPs become

360

embedded likely altering mobility of released metal, thus, reducing the exposure of root

361

epidermal cells to the released metal or NPs.

362

Increased transcript levels for metal stress are observed in the shoot tissues

363

(Anderson, unpublished). The shoots are not in immediate contact with the bacterium

364

and lack NPs on their surfaces.81 Also, transcripts, anticipated to have a role in drought

365

and pathogen stress, are elevated in the shoot tissues (Anderson et al. unpublished),

366

illustrating cross-talk between stress responses for metals, pathogen attack and water

367

deficiency. Annotation of the function of the transcripts indicates that many are 17 ACS Paragon Plus Environment


Page 18 of 48

368

associated with improved tolerance to water stress regulated by abscisic acid. This

369

finding indicates the NPs target another plant cell signaling process in addition to

370

modification of IAA. Preliminary studies with drought stress on plants reveals that PcO6-

371

colonized wheat seedlings growing with CuO NPs have a superior shoot stand after a

372

drought period than control plants grown without these treatments (Fig. 6). The

373

underlying mechanisms are being investigated and may relate to NP-induced cell wall

374

modifications as well as the altered transcriptome associated with drought tolerance.

375

The findings another consequence for the plant to exposure to NPs: the induction of a

376

higher level of plant tolerance to drought could be valuable under field conditions.

377

378

4. SUMMARY OF RHIZOSPHERE EFFECTS OF CuO AND ZnO NPs.

379

CuO and ZnO NPs alter processes important in the rhizosphere that are involved in

380

plant health in addition to the nutritional effect of increased tissue levels of Cu and Zn

381

(Fig. 6). These changes are correlated with specific effects on cell signaling

382

compounds that communicate between the plant and the root-colonizing microbe. The

383

consequences of NP challenge of the probiotic PcO6 involve altered levels of

384

interkingdom signaling metabolites including antifungal products (e.g., phenazines and

385

siderophores), quorum sensing coordinators (e.g., AHSLs), Fe-chelating siderophores

386

(e.g., pyoverdine) and the plant growth regulator IAA, Fig. 6). Thus, there is potential to

387

affect antifungal protection and Fe supply in the rhizosphere, as well as induced

388

systemic resistance in the plant.

389 390

Growth morphology in the plant may be altered by effects on the microbial levels of IAA as well as by direct disturbance of the flux of plant-produced IAA at the root tip. 18 ACS Paragon Plus Environment

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391

Increased lateral root formation (ZnO NPs) and proliferation of elongated root hairs

392

(CuO NPs) would have a beneficial impact on plant nutrition and water uptake. Also

393

there would be greater surface area for colonization by microbes. Systemic effects

394

induced by the NPs include cell signaling to promote altered gene expression increasing

395

the plants’ resilience to drought stress. The differences in the chemistry of the metals in

396

the metal oxide NPs governs the plant and microbial responses. Collectively the

397

findings illustrate the power of localized concentrations of the essential metals, Cu and

398

Zn, to affect rhizosphere processes that are key to plant health under environmental

399

stress.

400 401

5. FACTORS GOVERNING THE EFFICACY OF FORMULATIONS OF NPS FOR AGRICULTURE.

402

The basic scientific findings discussed for the CuO and ZnO NPs are from studies with

403

particles that were “as-made” from the manufacturer. Thus, any impact of engineered

404

coatings of the NPs that could alter their bioactivity is not explored in these studies. The

405

plants were grown without gross pore water movement, such as would result from a rain

406

event. The growth matrix, sand, was selected to reduce factors that could chelate

407

released metal ions. It lacked minerals, organic materials and other microbes

408

characteristic of native soil. The studies also are of a model system with only a single

409

microbe being addressed, whereas crop plants associate with diverse microbes, both as

410

endophytes and as surface colonizers. Microbial soil populations are diverse144,145 and

411

respond to many inputs including current farming practices that change plant diversity

412

and ecotypes.146-148The rhizosphere in agricultural soils has many microbial and

413

eukaryotic interactive partners that will influence responses to NPs (Fig 1).



414

Page 20 of 48

Consistent with the findings reviewed here and elsewhere is the importance of

415

the cell’s response to a gradient of metal release from the metal oxide NPs. Dissolution

416

of the metal oxide NPs could be intense at the site of the nanoparticle at an intracellular

417

or extracellular location with the cell.149 Soil factors especially pH would influence NP

418

dissolution. For example, acid soils would cause greater metal dissolution than a basic

419

soil.150 Microsites of different pH of the root surface exists; root hairs secrete protons

420

along with the plant metabolites. NP dissolution is promoted by metal chelators,

421

including siderophores, as well as the organic and amino acids that are part of root and

422

microbial exudates.46,151These acids would have the highest concentrations as they are

423

being released into the rhizosphere space, but metabolite levels will drop with distance

424

from the root surface in part through consumption by rhizosphere microbes.70 The

425

extent and composition of organic soil matter, such as the fulvic and humic acids, also

426

would affect metal bioavailability.151 The chemistry and sorption properties of the soil

427

would modify NP effects.152 Salts with Na, K and Ca as cations, at levels similar to those

428

in saline soils, relieve NP phytotoxic effects.153

429

A major research thrust with nanoagrochemicals is to boost the bioavailability of

430

the native soil. It is an innovative research field. Two recent papers show NPs increase

431

P supply to plants. In one paper, where ZnO NPs increase P uptake in mung bean, the

432

proposed mechanism involves a chemical “hand-off chain”. Enhanced Zn bioavailability

433

to the plant from the NPs allowed the roots to increase phosphatase activity which

434

liberated Zn from zinc phosphate generated the metal release from NP dissolution.154

435

The second paper, showing increased phosphate in lettuce upon growth with TiO2 and

436

Fe3O4 NPs, implicates several mechanisms, including NP- induced changes in root


Page 21 of 48


437

exudation that acidified the rhizosphere to enhance P release.155 A third paper reports

438

that Fe could be increased for rice grown in calcareous soils by the presence of coated

439

Fe3O4 NPs. The proposed mechanism involved Fe release from the NPs that

440

counteracted the effect on rice of excess Ca ions.156 Other nanosized products are cited

441

for improving plant resistance to pathogens. Such products contain silica157 and elicitors

442

of plant resistance, nano-sized glucans and chitosan.158,159

443

Engineering the formulations of the NPs by coating and mixing of chemical

444

composition will be required to optimize field performance. The formulations will differ

445

whether the products are applied to seeds, to the soil or as an aerial application. The

446

products may be time-released and targeted only to provide the stimulus at the right

447

location and the right time for the plant.18 Our abilities to predict the function of NP

448

formulations in different soils and with varied crops yet awaits the acquisition of

449

information bases to coordinate multiple physical, chemical and biological factors.

450

Acknowledgements. The authors thank the support of the USDA (NIFA Grant Number

451

10867118, 2012-2015 and funding to JEM from the Utah Water Research Laboratory.

452

We thank the assistance of many undergraduates and high school students who have

453

been engaged in this work: Joshua Adams, Alicia Calder, Stephanie Doxey, Jordan

454

Goodman, Trevor Hanson, Nicole Martineau, Jacob Stewart, Hannah Wagner, Melanie

455

Wright, Zac Zabrieski, Tommy Fang, Jean-Luc Watson, Andre Watson, and Dema Al

456

Qassy.

457 458 459



460

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Figure Legends

974

Fig. 1. The fertilizer potential of CuO and ZnO NPs. The essential metals, Cu and Zn,

975

are bioavailable to the plant from their metal oxide NPs through processes in the

976

rhizosphere influenced by soil properties, the root and its microbiome. Inserts show the

977

biofilm that forms over a wheat root surface by cells of a probiotic, Pseudomonas

978

chlororaphis O6; NPs are embedded within the biofilm (SEM large image). In the

979

absence of root colonization, association of CuO NPs with root hairs on the wheat

980

seedling root is revealed (light microscopy small image).

981

Fig. 2. Metal oxide NP uses in agriculture and factors that will need to be addressed to

982

optimize their efficacy in the rhizosphere for agricultural applications.

983

Fig. 3. Key rhizosphere metabolites produced by the probiotic, root colonizer,

984

Pseudomonas chlororaphis O6 (PcO6). The metabolites include acyl

985

homoserine lactones (AHSLs) that are inter- and intra-cellular cross-kingdom signaling

986

compounds. In PcO6 they stimulate production of antifungal metabolites, a family of

987

phenazines that are multitasked in promoting plant health. The pyoverdine-like

988

siderophores increase rhizospheric iron bioavailability to microbe and the plant. The

989

plant growth regulator indole-3-acetic acid is produced from tryptophan by

990

(PcO6. AHSLs, phenazines and siderophores also prime plant defense

991

mechanisms. Growth on plate medium of orange-colored (PcO6 cells from the

992

biofilms coating the roots of a colonized wheat seedling is shown (large image), and on

993

medium where secreted products inhibit the radial growth of Fusarium proliferatum



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994

mycelia (small image). Sublethal doses of CuO and/or ZnO NPs modify the production

995

of each of the shown metabolites.

996

Fig. 4. Summary of changes in PcO6 metabolism elicited by CuO and ZnO NPs.

997

The chemistry of these NPs is important because they have opposing effects on some

998

of the metabolic pathways.

999

Fig. 5. Changes in root morphology triggered by growth with CuO or ZnO NPs. The

1000

bright green fluorescence in cells revealed in the confocal images of the wheat seedling

1001

root tips have accumulated nitric oxide (NO). NO is part of the cell signaling system

1002

required for root hair formation and is detected by interaction with 4-amino-5

1003

methylamino- 2, 7, difluorofluorescein diacetate (DAF-FM DA). The white arrows denote

1004

root hairs. The distance between root cap and root hair initiation is very short for the

1005

seedlings grown with CuO NPs (green arrow). The inset image depicts the intense

1006

shortening of wheat seedling roots with growth with CuO NPs in pH 4.8 soil soluble Cu

1007

in the rhizosphere solution is heightened because dissolution is enhanced by the proton

1008

concentration. Wheat seedling growth with ZnO NPs results in increased lateral root

1009

formation.

1010

Fig. 6. The exposure of rhizosphere microbes and roots to CuO or ZnO NPs has

1011

outcomes in both the plant and root-associated microbe (PcO6). Nanoparticle-

1012

induced changes in the microbe would affect antagonism of fungal growth, plant root

1013

morphology, metal uptake into the shoots and plant responses to drought stress. Each

1014

of these responses has implications for plant health.

1015


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1016

1017

1018

TOC Graphic

1019 1020

Plant roots in agricultural soils are colonized with microbes, many of which enhance

1021

plant stress resilience. Pseudomonas chlororaphis O6 (PcO6) (atomic

1022

force microscope image of cells) has such beneficial properties. This microbe

1023

repurposes the C and N metabolites released from plant roots producing antibiotics,

1024

metal chelators, and plant growth regulators. These rhizosphere metabolites stimulate

1025

plant wellbeing. Biofilms of the bacterial cells on the root surface provide physical

1026

protection and hydration to the root. Growth with ZnO or CuO NPs modifies lateral root

1027

or root hair production at the tips, enhancing surface area for microbial colonization. The

1028

CuO and ZnO NPs differentially alter production of the microbial interkingdom signaling



Page 42 of 48

1029

metabolites. Thus, in addition to their ability to increase Cu and Zn levels in the plant as

1030

fertilizers, there may be other consequences of formulations containing CuO or ZnO

1031

NPs that can be tuned to benefit plant productivity in agricultural soils.

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CuO and ZnO Nanoparticles Modify Interkingdom Cell Signaling

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