Rhizosphere engineering: Enhancing sustainable plant ecosystem productivity Academic research paper on "Biological sciences"

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Rhizosphere Engineering: Enhancing Sustainable Plant Ecosystem Productivity in a Challenging Climate

Amirhossein Ahkami, Richard Allen White, Pubudu P Handakumbura, Christer Jansson

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PII: S2452-2198(17)30045-9

DOI: http://dx.doi.Org/10.1016/j.rhisph.2017.04.012

Reference: RHISPH56

To appear in: Rhizosphere

Received date: 28 February 2017 Revised date: 20 April 2017 Accepted date: 20 April 2017

Cite this article as: Amirhossein Ahkami, Richard Allen White, Pubudu I Handakumbura and Christer Jansson, Rhizosphere Engineering: Enhancin; Sustainable Plant Ecosystem Productivity in a Challenging Climate, Rhizosphere http://dx.doi.org/10.10167j.rhisph.2017.04.012

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Rhizosphere Engineering: Enhancing Sustainable Plant Ecosystem Productivity in a Challenging Climate

Amirhossein Ahkami 1*, Richard Allen White III 2, Pubudu P Handakumbura 1, Christer

Jansson 1*

1Environmental Molecular Science Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL), Richland, Washington 99352, USA

2Earth and Biological Sciences Directorate, Computational Biology, PNNL, Richland,

Washington 99352, USA

amir.ahkami@pnnl.gov

christer.jansson@pnnl.gov

Correspondence: Amirhossein Ahkami and Christer Jansson, EMSL-PNNL, Richland, Washington 99352, USA.

Abstract

The rhizosphere is arguably the most complex microbial habitat on earth, comprising an integrated network of plant roots, soil and a diverse microbial consortium of bacteria, archaea, viruses, and microeukaryotes. Understanding, predicting and controlling the structure and function of the rhizosphere will allow us to harness plant-microbe interactions and other rhizosphere activities as a means to increase or restore plant ecosystem productivity, improve plant responses to a wide range of environmental perturbations, and mitigate effects of climate change by designing ecosystems for long-term soil carbon storage. Here, we review critical knowledge gaps in rhizosphere

science, and how mechanistic understanding of rhizosphere interactions can be leveraged in rhizosphere engineering efforts with the goal of maintaining sustainable plant ecosystem services for food and bioenergy production in an ever changing global climate.

Keywords: Abiotic stress, Plant-Microbe Interactions, Plant Growth-Promoting Rhizobacteria (PGPR), Rhizosphere, Rhizosphere Engineering

1. Introduction

In the narrow zone of contact between soil particles and roots, the rhizosphere constitutes the first plant-influenced habitat encountered by soil microorganisms (Dessaux et al., 2016; Walker et al., 2011). Within this zone of plant-soil interaction, the rhizosphere is a dynamic and densely populated soil area with a complex set of inter-and intraspecies communications and food web interactions that significantly impacts carbon flow and transformation (Figure 1). The rhizosphere has been described to include three zones: the endorhizosphere, as portions of the root cortex and endodermis where microbes and mineral ions reside in the apoplastic space between cells; the rhizoplane, as the middle zone next to the root epidermal cells and mucilage; and the ectorhizosphere, as the outermost zone which extends from the rhizoplane out into the bulk soil (McNear Jr., 2013). It is important not to consider the rhizosphere as a region of definable size or shape, but rather as a gradient of chemical, biological and physical properties along the root (McNear Jr., 2013). The rhizosphere is strongly influenced by plant metabolism through the release of carbon dioxide (CO2) and secretion of photosynthate as an array of root exudates (mainly from the rhizoplane and

ectorhizosphere). Root exudates facilitate rhizosphere interactions by serving as energy sources for microorganisms and acting as chemical attractants and repellents (Bais et al., 2001; Estabrook and Yoder, 1998). They serve as communicating molecules to initiate biological and physiological interactions between the soil microbiome and the plant roots by influencing the chemical and physical properties of the soil and the soil microbial community, inhibiting growth of competing plant species, facilitating beneficial symbioses, e.g. with nitrogen-fixing bacteria, mycorrhizal fungi and epiphytes, and by preventing pathogenic bacterial, fungal and insect attacks (Nardi et al., 2000).

The rhizosphere is of paramount importance for ecosystem services, such as carbon and water cycling, nutrient trapping, crop production, and carbon uptake and storage (Adl, 2016). Global climate change, including rising temperatures and disruptive weather patterns due to increasing levels of atmospheric CO2, will affect rhizosphere ecology, and hence ecosystem function, through a variety of direct and indirect ways. For example, it has been estimated that increasing global temperatures between 1981 and 2002 reduced the yields of major cereals by almost $5 billion per year (Lobell and Field, 2007). Abiotic stresses such as drought, high temperatures and salinity are major causes for loss of natural vegetation, crop productivity depreciation and, therefore, reduction of capacity for CO2 uptake. Drought stress severely inhibits photosynthesis and root growth (Iturbe-Ormaetxe et al., 1998; Verslues, 2017). Salinity stress leads to ion toxicity due to excessive amounts of Na+ and Cl- that causes detrimental effects on plant growth and development (Ashraf, 1994; Ashraf and Khanum, 1997; Negrao et al., 2017). Drought and salinity stresses both cause elevated levels of ethylene, which is

inhibitory to root growth and therefore affect a number of plant physiological pathways (Belimov et al., 2002; Sun et al., 2007). Additional environmental stresses negatively impact plant growth and development in a number of ways like disturbing hormone balance and increasing susceptibility to diseases (Ashraf, 2003; Glick et al., 2007; Saleem et al., 2007).

Plant survival under abiotic stress conditions requires extensive physiological adaptations. In this regard, plant hormones play crucial roles in the process of root formation and growth and in regulation of root morphological responses to abiotic stress. Hormone perception and crosstalk create an intricate network in which abiotic stresses can interfere, resulting in root growth alterations in the rhizosphere. The phytohormone auxin is the key regulator for almost all aspects of growth and development in plants. In roots, the most important auxin-associated phenotypes are the increase in the length of root hairs, the bimodal effect of auxin concentration on primary root length, the dose-dependent increase in number of lateral root primordia, and the response to gravity (Overvoorde et al. 2010; Pitts et al. 1998; Rahman et al. 2002; Ishida et al. 2008; Peret et al. 2009). Other types of plant hormones like cytokinins and gibberellic acids (GAs) act as negative regulators of root development and positive stimulators of root elongation, respectively. It was shown that exogenous cytokinin treatment inhibits root elongation (Beemster et al., 1998), while reduction of endogenous cytokinin levels increases primary root elongation (Werner et al., 2003; Miyawaki et al., 2006). The involvement of abscisic acid (ABA) in responses to abiotic stresses and in particular to drought is well characterized. When the roots sense a

decrease in soil water in the rhizosphere region, first ABA biosynthesis in the root tips is increased and then it is transported to the leaves leading to induction of stomatal closure. It was reported that primary root elongation is inhibited by ABA during drought stress (Sauter et al., 2001; Smith and Smet, 2012; Xiong et al., 2012). Ethylene is a gaseous plant hormone that has significant influences on many aspects of plant growth and development. Ethylene is a strong inhibitor of root elongation and stimulates root hair formation (Negi et al., 2008). Strigolactones and their derivatives were recently defined as novel phytohormones that play important roles in shoot and root growth. In particular, Strigolactones and their derivatives promote the elongation of primary roots and adventitious roots, while repressing lateral root formation (Sun et al., 2016). Also, it should be noted that hormonal crosstalk is essential for plant growth and development. For example, ethylene controls root formation by regulating auxin transport within the root tip (Swarup et al., 2007), and stimulates the expression of auxin biosynthesis genes, and of AUX1 (auxin transporter protein 1) and PIN2 (PIN FORMED2 auxin transporter), resulting in an increased basipetal auxin transport. Plant phytohormones are also important in the context of beneficial plant-microbe interactions (see below). Therefore, plant phytohormones should be considered as one of the key factors in all efforts dealing with rhizosphere engineering (Fig. 1 and Fig. 2).

While plants may acclimate to abiotic stress tolerance through phenotypic plasticity, associations with naturally occurring microorganisms provides another means for enhanced resistance to, or protection from, various abiotic stresses (Farrar et al., 2014). The microbiota or microbiomes of plants, like those of humans and many other

eukaryotes, comprise a staggering diversity of microorganisms inside and outside their tissues, in the endosphere and ectosphere, respectively (Berg et al., 2014; Schlaeppi and Bulgarelli, 2015; Vandenkoornhuyse et al., 2015). Due to their intimate association with microbes, plants could be considered meta-organisms, or holobionts, between the plant per se and its interacting microbiota (Vandenkoornhuyse et al., 2015), and the genome of the plant microbiome is sometimes referred to as the second genome of the plant. The plant-microbe interactome covers a spectrum of associations ranging from mutualistic, commensalistic and parasitic relationships and represents an ancient co-evolution (Heckman et al., 2001). A classical example of ancient symbiosis in plants is with arbuscular mycorrhizal (AM) being the oldest, over 400 million years (Heckman et al., 2001). Of paramount interest are the interactions between plants and the rhizospheric microbial communities (Philippot et al., 2013). This complex plant-associated microbial community is critical for plant health and productivity (Berendsen et al., 2012). As is further detailed below, many isolated bacterial strains have been identified as plant growth-promoting bacteria (PGPB), which can stimulate plant growth through a number of direct and indirect mechanisms (Kloepper and Schroth, 1981). Such mechanisms include nutrient solubilization, biological nitrogen fixation, induction of systemic resistance, production of plant growth regulators, organic acids, and volatile organic compounds (VOCs) as well as protection by enzymes like 1-aminocyclopropane-1-carboxylate (ACC)-deaminase, chitinase and glucanase.

Plant Growth-Promoting Rhizobacteria (PGPR) are a group of PGPBs inhabiting the proximity or surface of the roots and are involved in promoting plant growth and

development by direct release of microbial exudates (e.g., metabolites and small peptides/lipids) in the vicinity of rhizosphere (Kloepper and Schroth, 1981). The first use of the word "PGPR," was by Kloepper and Schroth (1978), but soil microbes influencing plant growth has been known for over one hundred years (Hartmann et al., 2008). PGPR have been shown to increase productivity in a variety of different crops (e.g., tomato, lettuce, wheat, soybean, rice and apples) under normal and stressful conditions (Deshmukh et al., 2016; Etesami and Alikhani, 2016; Bhattacharyya and Jha, 2012). Their mode of action includes broad-spectrum antagonism in biocontrol of soil-borne pathogens (Nakkeeran and Fernando, 2005), immobilization of nutrients (e.g. phosphorus) (Bhattacharyya and Jha, 2012), release of plant hormones (Vessey, 2003), and direct fixation of nitrogen (e.g. bradyrhizobium and rhizobium) (Zahran, 2001). Axenic cultures exhibiting PGPR effects have been obtained from hundreds of rhizospheric isolates (Bhattacharyya and Jha, 2012). However, a vast majority of these PGPRs are not amenable to cultivation, or exists as co-culture dependents.

The physical and chemical context of the rhizosphere is the result of many competing and interacting processes that depend on soil type and water content, the composition of microbial communities, and the physiology of the plant itself (Ryan et al., 2009). For better plant productivity, all three components of the rhizosphere, plant, soil and microbes can be engineered. The soil can be amended or managed to improve its overall quality by changing its physical and chemical properties, microbiomes can be selected for beneficial traits such as promoting plant growth and root characteristics, and the plants can be engineered to harbor beneficial and novel traits of interest.

Natural and synthetic plant-microbe interactions can be utilized to improve nutrient bioavailability. The key chemical compounds used in the plant root-microbe communications can be used as targets for genetic engineering to further enhance these interactions. The present review mainly focuses on plant and microbe (PGPRs) components in rhizosphere engineering.

2. Harnessing Natural and Beneficial Plant-Microbe Interaction in the Rhizosphere Region

Various strategies have been described to alleviate stress-induced adverse effects on plant growth. Transcriptome engineering is a promising approach for generating abiotic stress-tolerant crops, and to date, constitutive overexpression of single genes encoding enzymes related to the accumulation of osmolytes and proteins that function as reactive oxygen species (ROS) scavengers and ion transporters has been the most common strategy for improving abiotic stress tolerance in plants (Reguera et al., 2012). Nevertheless, due to involvement of multiple pathways in plant acclimation to stress and possible pleiotropic effects on plant growth (Ori et al., 1999; Rivero et al., 2007), this approach has met with limited success. Application of agrochemicals (i.e. pesticides) is another approach in boosting crop productivity but is contentious due to their cost and environmental concerns about their long-term utilization in soil. An alternative strategy to mitigate climate change impacts on plant fitness is the utilization of beneficial mutualistic plant-microorganism interactions in the rhizosphere. Such mutulism can offer enhancement of root nutrient uptake and biomass

productivity, and potentially improved plant acclimation to abiotic stresses (Mirshad and Puthur, 2017; Vurukonda et al., 2016b). Identification of rhizospheric microbes capable of conferring stress tolerance to their plant hosts, and employing symbiont-based approaches to understanding and improving root biomass production, whole plant productivity and/or soil carbon storage, could significantly contribute to reducing the negative impact of abiotic stresses on plant ecosystem function. There are several advantages to this approach, including the capability of PGPRs in conferring more than one type of abiotic stress tolerance (e.g. through affecting plant hormone pathways), and the applicability to a wide variety of diverse plant hosts (Coleman-Derr and Tringe, 2014). Recently, PGPRs have received substantial attention for their ability to confer benefits to crop productivity and stress tolerance, similar to what have been achieved through time-consuming plant breeding programs (Barrow et al., 2008; Marasco et al., 2013; Marulanda et al., 2009; Mayak et al., 2004; Tank and Saraf, 2010). For example, the growth-promoting bacterial, Burkholderia phytofirmans strain PsJN, has been shown to colonize and promote growth of switchgrass under different conditions, especially in the early growth stages (Kim et al., 2012), suggesting it may be a promising candidate for improving bioenergy crop production. Strain PsJN can significantly stimulated growth of specific genotypes of switchgrass under sub-optimal conditions, pointing the way to the development of a sustainable feedstock production system (Kim et al., 2012).

2.1. Drought

Drought is the single most critical threat to plant and crop productivity; it impairs plant growth and development more than any other environmental factor (Anjum et al.,

2011; Harb et al., 2010; Shao et al., 2009). Drought stress is affected by climatic, edaphic and agronomic factors, and in view of climate change and limiting global freshwater supply it has been predicted that the impacts of drought will be further aggravated in the future (Farooq et al., 2009; Somerville and Briscoe, 2001). With forecasted global changes in temperature and precipitation, drought will increasingly impose a challenge not only to food and feed but also to biomass production (Ings et al., 2013; Quinn et al., 2015; Yin et al., 2014). Most of the bioenergy crops have some degree of drought susceptibility as revealed for example through measures of low water-use efficiency (WUE) in Miscanthus (Ings et al., 2013; Yin et al., 2014) and poplar (Viger M et al., 2013). Thus, in addition to the urgent need of developing drought-tolerant crops for food security (Lopes et al., 2011), it becomes imperative to improve drought tolerance and WUE in bioenergy crop plantations for sustainable biomass production in arid and semi-arid regions (Ings et al., 2013; Quinn et al., 2015; Yin et al., 2014).

Despite an extensive volume of scientific reports on enhancing plant drought tolerance using a variety of genetic engineering approaches, progress has been slow, demonstrating the complexity of the trait and the large number of genes involved. The rhizosphere and associated microbiota play critical roles in controlling the ability of plants and plant ecosystems to cope with drought. PGPRs colonize the rhizosphere/endo-rhizosphere of plants and confer drought tolerance by: (i) producing exopolysaccharides (EPS), 1 -aminocyclopropane-1 -carboxylate (ACC) deaminase, VOCs, phytohormones like abscisic acid (ABA), gibberellic acid, cytokinins, and indole-3-acetic acid (IAA); (ii) inducing accumulation of osmolytes and antioxidants; and (iii)

regulation of stress-responsive genes and alteration in root morphology (Vurukonda et al., 2016a). For example, IAA-producing Azospirillum spp. improved tolerance to drought stress in wheat by enhancing root growth and formation of lateral roots (Arzanesh et al., 2011). Similarly, PGPR Bacillus thuringiensis stimulated the growth of a lavender species (Lavandula dentate) under drought conditions due to the production of IAA, which improved nutrition, physiology, and metabolic activities of the plant (Armada et al., 2014). In the same vein, Rolli et al. (2015) recently demonstrated the contribution of GFP-labelled Acinetobacter spp. and Pseudomonas spp. isolates in improving acclimation to drought in Arabidopsis and grapevine through a water stress-induced mechanism. Leaves of oriental thuja (Platycladus orientalis) inoculated with Bacillus subtilis increased stomatal conductance and ABA levels in shoots, conferring drought resistance to container-grown plants. Inoculated plants had increased leaf-water content and water potential and an increase in cytokinin concentration, which was linked to the increase in ABA levels (Liu et al., 2013). In another study, Phyllobacterium brassicacearum strain STM196, isolated from the rhizosphere of oilseed rape (Brassica napus), was shown to help alleviate drought stress in inoculated Arabidopsis plants by increasing ABA levels and decreasing leaf transpiration, thereby enhancing osmotic stress tolerance (Bresson et al., 2013). Also, soybean plants inoculated with gibberellin-producing rhizobacterium Pseudomonas putida strain H-2-3, were found to show enhanced shoot length and fresh weight under drought stress. These soybean plants also accumulated higher levels of chlorophylls, salicylic acid and abscisic acid in response to stress, compared to control plants (Kang et al., 2014b). In a promising approach by Timmusk et al. (2014), soil microbial communities were isolated from harsh

environments and used to prime wheat plants. Out of a dozen or more isolates, Bacillus thuringiensis strain AZP2 and Paenibacillus polymyxa strain B were able to confer improved drought tolerance to wheat seedlings (Timmusk et al., 2014).

2.2. Salinity

Salinity is a major environmental stress that severely affects plant productivity throughout the world. Excess salt triggers ion toxicity and ion imbalances in plants, thus inducing metabolic imbalances and hyperosmotic stress-induced water deficit. Plants cope with salinity stress by synthesizing osmolytes and polyamines, by activating defense mechanisms, reducing ROS accumulation, ion transport, and compartmentalization. In a study by Upadhyay et al. (2011), inoculated wheat seedlings with EPS-producing PGPRs (including Bacillus spp. Enterobacter spp. Paenibacillus spp.) showed significantly decreased Na+ uptake and increased biomass production under high-saline conditions. Also, inoculated tomato plants with certain PGPRs were shown to be able to maintain their growth under high-salinity and water-limited conditions by reducing the negative impact of stress-induced ethylene release on root growth through the activity of bacterial ACC-deaminase (Mayak et al., 2004). In a recent study, Bharti et al. (2016) demonstrated the use of a carotenoid-producing halotolerant PGPR Dietzia natronolimnaea strain STR1 in conferring salinity tolerance in wheat. Inoculated plants had higher expression of proline and various antioxidants, enabling these plants to withstand salinity stress (Bharti et al., 2016). Moreover, ABA signaling, SOS pathways and iron transport were activated in these plants as a result of PGPR

inoculation. Similarly, five bacterial isolates belonging to the genera Klebsiella, Pseudomonas, Agrobacterium, and Ochrobactrum were able to confer salt tolerance in peanut seedlings. Under salinity stress, peanut seedlings were able to maintain ion homeostasis, had less ROS buildup with enhanced growth, compared to non-inoculated seedlings (Sharma et al., 2016). Another study by Mahmood et al., (2016) demonstrated that Enterobacter cloacae and Bacillus drentensis acted synergistically with foliar application of silicon to confer salinity tolerance in field-grown Mung beans. Moreover, Brachybacterium saurashtrense strain JG-06, Brevibacterium casei strain JG-08, and Haererohalobacter spp. strain JG-11 conferred improved plant growth in peanuts when seedlings were subjected to salinity stress by adding 100M NaCl. Plant height, shoot length, root length, shoot dry weight, root dry weight, and total biomass were significantly higher in inoculated plants compared to uninoculated plants (Shukla et al., 2012).

2.3. Elevated CO2 Levels

Plants take up CO2 from the atmosphere through photosynthesis and store it as organic carbon in plant sinks, or in microbial biomass or the soil via allocation to the roots. Enhanced C3 photosynthesis caused by rising atmospheric CO2 levels may stimulate rhizospheric microbes by increased transfer of photosynthate to the soil. A more long-term effect on the soil and soil microbes emanates from climate-induced shifts in plant diversity and composition, which, in turn, will alter the quantity and quality of soil organic material. It is clear that both these climate change-induced and plant-mediated effects on soil properties and soil biota will have major impacts on carbon

cycling, uptake of methane and nitrous oxide, and climate in terrestrial ecosystems (Bardgett, 2011; Ostle et al., 2009).

Generally, utilization of PGPRs has turned up as a technology to facilitate grassland management (van der Heijden et al., 2006), ecosystem restoration (Requena et al., 2001) and reforestation (Chanway, 1997). The potential of PGPRs for improving terrestrial carbon storage through increasing plant productivity and decreasing microbial respiratory carbon loss under rising atmospheric CO2 condition has been described (Nie et al., 2015). Thus, it is possible that elevated atmospheric CO2 levels under future climate scenarios will increase the dominance of PGPRs. How these plant-microbe interactions are established, how the productivity of a host plant is influenced by its microbiome, and how particular microbiomes alleviate plant stress is largely unknown. Therefore, not only mechanisms responsible for plant growth promotion have to be thoroughly investigated, but also a detailed understanding of all steps involved in plant colonization by bacteria is required in order to reap the maximum benefits of PGPB in improving plant ecosystem performance.

2.4. Temperature

High temperature (e.g., heat shock) is a critical abiotic environmental challenge to both plant and microbial growth and homeostasis. Finding PGPR isolates that allow for plant growth at higher temperatures would potentially expand the global range of crop cultivation, particularly with reference to future climate scenarios. Experimental approaches with inoculation of PGPR isolates that improve plant thermal stress are sorely lacking. To date, research has focused on selecting PGPR isolates that are

themselves heat tolerant up to 60 °C (e.g., rhizobial isolates) (Rodriguez et al., 2008), but not for isolates that confer heat tolerance in their host plant. However, some studies report the application of PGPRs to alleviate the side effects of low-temperature stress on plant growth (Ait Barka et al., 2006; Dimkpa et al., 2009). Physiological changes observed during plant cold acclimation or hardening include increases in sugar, proline and anthocyanin contents (Dimkpa et al., 2009). Grapevine plants inoculated with Bacillus phytofirmans strain PsJN accumulated higher levels of carbohydrates, proline, phenols, and showed elevated rates of photosynthesis and starch deposition compared to control plants during cold stress (Ait Barka et al., 2006). Moreover, inoculation of grapevine with the same PGPR strain lowered the rate of biomass reduction and electrolyte leakage (an indicator of cell membrane injury) during cold treatment at 4 °C, and also promoted post-chilling recovery.

2.5. Heavy metals

Heavy metals such as zinc (Zn), arsenic (As), chromium (Cr), cadmium (Cd), mercury (Hg), copper (Cu), nickel (Ni), and lead (Pb) represent a significant challenge to plant and microbial growth if elevated above tolerance levels and/or above essential needs (Cu, Ni, Cr, Zn are essential at lower levels). Plants and their phytoremediation potentials are usually influenced by the toxic effects of metals in soil; however, application of soil bacteria can enhance phytoremediation, hence the term microbe-assisted phytoremediation (Glick, 2003; Jamil et al., 2014).

Upon inoculation, many PGPRs have demonstrated the ability to protect their host plants from heavy-metal toxicity (Shinwari et al., 2015). Genera of PGPRs shown to have this ability cover a wide diversity from the Alphaproteobacteria (e.g., the genera Mesorhizobium, rhizobium, Sinorhizobium, Bradyrhizobium), Betaproteobacteria (e.g., Achromobacter), Y-proteobacteria (e.g., Azotobacter, Pseudomonas) to Firmicutes (e.g., Bacillus spp. many strains) (Shinwari et al., 2015). For example, an inoculation of Bacillus licheniformis strain NCCP-59 improved seed germination and biochemical attributes of rice under Ni stress suggesting the ability of this strain to protect the plants from the toxic effects of heavy metals (Jamil et al., 2014).

PGPRs, like most microbes have evolved unique ways to use, transform, mobilize and immobilize heavy metals to make them tolerable, or even utilizable, or render them inactive. These scenarios involve five main mechanisms: (1) Extrusion by transport via efflux pumps; (2) Exclusion by removing metals from target sites; (3) Inactivation by complexation (e.g., forming thiol-containing molecules); (4) Biotransformation to a less toxic redox state (e.g., Cr+4 vs. Cr+6); and (5) Methylation and demethylation (Tak et al., 2013). Plants also exhibit a variety of mechanisms for heavy metal resistance; however, the relationship between the microbial and plant processes for heavy metal resistance at the molecular level is unclear. Furthering our understanding of plant-microbe interactions and the genes and regulatory mechanisms responsible for heavy metal resistance will improve our ability to engineer plants for growth on contaminated sites, for production as well as for spill cleanup.

3. Molecular Mechanisms of Symbiotic Plant-Microbe Interactions

The ability of PGBRs to improve plant growth under stressful conditions is well documented in the literature (Bhattacharyya and Jha, 2012; Coleman-Derr and Tringe, 2014; Goh et al., 2013). Although substantial advances in elucidating the genetic basis for the beneficial effects of PGPRs on plants have been made, e.g. from whole-genome sequencing projects (Bloemberg and Lugtenberg, 2001), few publications are available that explore the molecular basis behind plant growth promotion by rhizobacteria (Table 1). Screening the mechanisms underpinning activities by PGPR would pave the way for genome-editing efforts on plants and microbes for the purpose of enhancing plant growth, particularly in stressful environments.

In this context, Wang et al. (2005), performed microarray analysis to gain insights into physiological and biochemical changes in the host plant Arabidopsis inoculated with the PGPR Pseudomonas fluorescens strain FPT9601-T5. The results showed that approximately 200 out of 22,810 Arabidopsis genes could be identified as differently expressed (with >2 fold change) in bacteria-treated plants. The majority of these genes fall into functional categories, such as basic metabolism, signal transduction, and stress response. Specifically, the findings showed that upon PGPR colonization, putative auxin-regulated genes and nodulin-like genes were up regulated, and some ethylene-responsive genes were down regulated (Wang et al. 2005). Vargas et al. (2014), using Illumina RNA-Seq technology, showed that bacterial inoculation with Gluconacetobacter diazotrophicus strain PAL5 activated ABA-dependent signaling genes conferring drought resistance in sugar cane. Kim et al. (2015) demonstrated that the VOCs from

Bacillus subtilis strain JS affected gene expression profiles in tobacco. Tobacco genes related to photosynthesis pathways were up regulated in plants inoculated with the PGPR strain, suggesting a VOC-mediated enhancement of plant growth (Kim et al., 2015).

Besides the above-mentioned investigations on gene expression profiles, proteomic analyses will provide additional information on the protein(s) and pathways induced during host-PGPR interaction. Identification of candidate proteins in various types of PGPRs will provide valuable resources for growth promotion targets in further research (Walters et al., 2005). The Bacillus amyloliquefaciens strain KPS46 was investigated for its ability to activate extracellular protein elicitors for enhanced plant growth and induced systemic resistance in soybean plants (Buensanteai et al., 2008). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), mass spectrometry (MS) and protein database searching were used to separate extracellular proteins secreted by KPS46 wild type and N19G1, a mutant of KPS46 with reduced production of extracellular proteins and lacking growth promotion activity. The analysis revealed 20 extracellular proteins that might be involved in plant growth promotion and induced resistance (Buensanteai et al., 2008). Also, screening for molecular effects induced in pepper-PGPR interactions under drought-stress conditions led to identification of six differentially expressed stress proteins in pepper plants inoculated with Bacillus licheniformis strain K11 (Lim and Kim, 2013). Although there are some constraints for using proteomic approaches in identifying proteins in PGPR-plant interactions, e.g. sample preparation issues and limited information available in protein databases (Schenk et al., 2012), advanced molecular biological techniques including MALDI-TOF

and top-down proteomics hold great promise for improved understanding of the molecular basis for plant-PGPRs interactions in the near future.

Metabolite profiling of both plant and bacteria is another approach to dissect molecular mechanism of symbiotic interactions. For example, using a GC-MS approach, Timmusk et al. (2014) monitored emissions of seven stress-related VOCs from rhizosphere bacterially-primed drought-stressed wheat seedlings, and demonstrated that three of these volatiles (i.e., benzaldehyde, p-pinene and geranyl acetone) are likely promising candidates for a rapid non-invasive technique to assess crop drought stress.

Knowing the genes/proteins/metabolites involved in PGPRs-plant interactions that are responsible for abiotic stress resistance may allow for creating engineered plants harboring genes that prevent stress, and/or microbes that could be used to alleviate stress (Figure 2).

4. Rhizosphere Design and Engineering

Plant ecosystems are valued for a variety of reasons, e.g., food, feed, and fuel productivity (and hence human livelihood and income), climate regulation, carbon and water cycling, carbon storage, nutrient trapping, provision of wildlife habitats, and recreational activities. Given the wide range of genotypes that can be collected and/or generated per a specific plant species, genetic diversity is a potentially important asset in maintaining or increasing plant ecosystem values, e.g., in controlling stability and stress resilience in native and cultivated ecosystems, productivity in cultivated ecosystems, and ecosystem function. This notion suggests that selection of both

genotypes and species should be considered in ecosystem design and breeding programs, particularly in taking advantage of, or mitigating potential negative effects of, changes in climate, as well as in avoiding adverse impacts on regional or global climate.

Designing or engineering plant ecosystems for enhanced carbon storage includes increasing carbon allocation to above-ground or below-ground biomass for partitioning into structural components, or transfer to the soil for conversion to recalcitrant minerals such as calcite or into soil aggregates. The potential for engineering plants with improved capacity in carbon storage was reviewed by Jansson et al. (2010), where they coined the term "phytosequestration". The wider perspective of using terrestrial ecosystems for carbon storage has been extensively discussed by Lal and coworkers (Lal, 2004, 2008a, b; Lal et al., 2007). For long-term carbon storage in the soil it becomes important to unravel details in the metabolism of rhizospheric microbial communities and their interactions with the plant host, and in mechanisms of soil carbon deposition.

4.1. Rhizosphere Engineering via Plant Biotechnology

Traditional plant breeding approaches and advanced plant genome editing-based methods are promising ways to accumulate favorable alleles associated with stress tolerance in a plant genome. One of the main ways plants modify the rhizosphere is through root exudation and there have been few attempts in this context to engineer the rhizosphere by manipulating the efflux of H+ and organic anions from the roots in transgenic plants (Ryan et al., 2009). Since many genes controlling exudates have been

identified, it seems feasible to modify expression levels of those genes in plants to redesign rhizosphere for improved features. For example, transgenic rice and tomato plants transformed with the Arabidopsis vacuolar H+-pyrophosphatase gene AVP1, showed approximately 50% greater citrate and malate efflux than wild-types when treated with AlPO4. This was interpreted as a means to enhance resistance to Al3+ stress and improve the ability to utilize insoluble phosphorus (Yang et al., 2007). However, it is important to note that plant engineering to impact rhizosphere could be a very complex process due to degradation or inactivation of the engineered compound in the soil, small rate of exudation to influence the rhizosphere, limited knowledge about root exudates composition, and changing of exudate releasing time and levels with plant development and external stimuli.

Understanding how to control photosynthate distribution in plants are key to being able to alter its allocation between aboveground and belowground biomass, as was demonstrated (e.g., in the "low-methane high-starch" rice project) (Su et al. 2015) and in work on carbohydrate transporters (Ryan et al. 2009). An alternative option is to explore genotypic diversity in selecting crops or native-ecosystem plants with desirable traits for carbon allocation and partitioning. It can be argued that increased allocation of photosynthate to roots and soil in the rhizosphere region will be at the expense of carbon partitioning into harvestable compounds like lignocellulosics or grain (i.e. partitioning efficiency or harvest index). However, there is ample evidence that photosynthesis is often sink limited and feedback inhibited by insufficient sink demand (Jansson et al., 2010; McCormick et al., 2006; Paul and Foyer, 2001; Paul and Pellny, 2003; Yadav et al., 2015). Thus the potential exists to steer an increased amount of

carbon to the belowground for long-term carbon storage without jeopardizing productivity.

4.2. Rhizosphere Engineering via Synthetic Rhizospheric Microbial Communities

Bioengineering of synthetic microbial communities for plant/crop growth promotion, disease resistance, and stress tolerance/regulation presents a unique opportunity. While hundreds of bacterial strains have been identified to have many beneficial effects; engineering a sustainable synthetic microbial community represents a significant challenge. For example, in a simple two strain co-culture, six ecological interaction factors must be taken into account (Grosskopf and Soyer, 2014): 1) Commensalism, that is one strain benefits from the other without affecting it, for example products from one strain serves as substrates for the other; 2) Competition, for example substrate competition; 3) Predation, that is the predator benefits while the prey is disadvantaged; 4) No interaction, a net zero effect with no shared substrates, no predator-prey relations, and no competition; 5) Cooperation, both strains benefit from each other, or syntrophy or cross-feeding: and 6) Amensalism, one strain is negatively affected while the other strains remain unaffected. The complexity of these possible ecological interactions will scale linearly with the addition of extra strains (Grosskopf and Soyer, 2014). The main challenges include minimizing parasitism and competition while maximizing beneficial effects and cooperation. Minimizing competition is particularly challenging as even in two-strain co-cultures competition tends to dominate rather quickly (Foster and Bell, 2012). Environmental conditions such as pH,

temperature, nutrient availability, and host plant exudates will also affect growth rates, seeding rate, stabilization, susceptibility to pathogens, and sustainability of the synthetic microbial community once applied.

Setting challenges aside, building a synthetic microbial community can leverage knowledge obtained from naturally occurring microbial communities containing PGPRs. Many microbial genera are known that colonize the rhizosphere, have publically available genome sequences, and are amenable to genetic engineering efforts. These genera include Pseudomonas (Rothmel et al., 1991), Bacillus (Dong and Zhang, 2014), Paenibacillus (Kim and Timmusk, 2013), Streptomyces (Medema et al., 2011), and Rhizobium (Patel and Sinha, 2011). While Streptomyces spp. offer great examples of PGPRs with tractable genetic systems and many available complete genome sequences, Streptomyces spp. have some drawbacks in that they have very large genomes (on average ~7 MB) (Koberl et al., 2015), and have many mobile elements, which make them difficult to engineer and/or grow in cooperative synthetic microbial communities. The base of a synthetic community would be Bacillus spp., as it is relatively easy to genetically engineer (Dong and Zhang, 2014), has a large depth of complete genome sequences (Sharma and Satyanarayana, 2013), contains many isolates that have plant growth proprieties (Koberl et al., 2013; Koberl et al., 2015), and/or are currently used in bio-control applications. For example, a Bacillus spp. could be engineered to contain a nitrogen-fixation machinery (e.g., NifH from Paenibacillus (Kim and Timmusk, 2013), produce high concentrations of plant hormones (Arkhipova et al., 2005), or add pathways from other Bacillus spp. to control pathogens (Koberl et al., 2013). Psuedomonas, Rhizobium and/or Bradyrhizobium genera could be added for

increased nitrogen fixation. A simple three-strain member consortium, including an engineered Bacillus spp. with two natural or engineered nitrogen fixers like Pseudomonas, Rhizobium and/or Bradyrhizobium, could provide many of the benefits of a more complex natural rhizosphere community. To add an element of control towards cooperation over competition, genes could be eliminated from each member such that synthesis of an essential co-factor would require the biosynthetic machinery of all strains. A conceivable scenario would be a system where the Bacillus needs a co-factor from the Pseudomonas, the Pseudomonas is dependent of an essential gene from the Rhizobium, and, finally, the Bacillus could remove a waste product produced by the Rhizobium that is subsequently recycled by the Bacillus for mutual use. The potential ecological functional interactions increase with the number of strains added; a three-strain consortium would potentially contain >729 predicted interactions, and a four-member consortium 531,441 predicted interactions (Grosskopf and Soyer, 2014). Efforts should be taken to limit the number of strains within a synthetic microbial community to three strains in order to exert control of potential interactions.

In designing a microbial community for an engineered rhizosphere, critical elements must be considered in the realm of microbial competence (Bashan et al., 2014). Several questions relating to microbial traits must be considered prior to selection of a microbe for building an engineered microbial consortium: (1) How proficient is the colonization of the microbe and target plant root surface in the rhizosphere? (2) Can this microbe colonize the target host plant effectively? (3) Can the microbe survive, grow and compete with the rest of the microbes in the consortia? (4)

Does the microbe attach well to host root surfaces? (5) Does the microbe promote plant growth or provide enhancement to plant growth promoting members of the consortia? (6) How does the strain handle abiotic stress such as drought? and (7) Does the microbe reach the required density of growth? (Calvo et al., 2014; Yang et al., 2008). Microbes that are motile and chemotactically driven to roots by the host's plant root exudates and with strong attachment to the host plant allows for stronger association and possibly better beneficial effects to the plant (Yang et al., 2008; Bashan et al., 2014). Density of growth is an important factor for whether or not the microbe will have a beneficial effect. An example of this is Pseudomonas spp., which needs a minimal growth density (i.e., 105-106 CFU g-1 of root) in order to protect plants from pathogens (namely G. tritici and Pythium spp.) (Hass and Defago, 2005). If standard agronomic farming practices are used then the microbial consortia must be able to tolerate herbicides, fertilizers, and pesticides without losing any of the beneficial effects.

Other challenges that remain once the proper microbes have been selected are how, when, and where to inoculate? For example, does inoculation work better during the day or night; should inoculation be applied to root, leaves, or both; and does the inoculum need to be alive or can it be lyophilized? Examples of inoculates most commonly applied directly on seeds (for improved crop performance) are bioprimed, film coated, slurry coated or pelleted (reviewed in O'Callaghan, 2016).

5. Conclusions and Future Opportunities

As a nation and as a world, we are faced with the grand challenge of navigating the needs for dramatic increases in crop yields, environmental stewardship, and

predicting the link between plant ecosystems and atmospheric processes, including the global effects of climate change (Furbank and Tester, 2011; Hetherington, 2013; Long and Zhu, 2009; Ort et al., 2015). To meet these taxing demands, we need to unleash the full potential of plant ecosystem productivity in diverse and stressful environments (Long et al., 2015; Long and Zhu, 2009; Ort et al., 2015), and build a knowledge base that enables the prognostic ability to predict, on one hand, how plant ecosystems, including the rhizosphere, are affected by climate change, and, on the other hand, how cycles of carbon, water, and nutrients in plant ecosystems influence climate (U.S. DOE Workshop Report, 2015). Here, the emerging field of rhizosphere and ecosystem engineering offers an exciting and powerful opportunity to fill critical knowledge gaps and provide solutions. For example, how the entirety of the ectophytic and endophytic microbial communities interacts within itself, including mycorrhizal-rhizospheric microbiome interactions (Linderman, 1988), and with the plant hosts in growth-promoting and growth-compromising activities remains largely unknown. Unraveling the plant-microbe interactome will be key to understanding and exploiting the full yield potential of a cropping system, and to understanding the mechanisms behind rhizosphere priming (Studer et al., 2016) and managing the carbon cycle in the soil under current and future climates. Future research programs will apply synthetic biology approaches, make use of favorable root-microbe interactions and employ a combination of both approaches to improve the productivity of major food and bioenergy crops under biotic and abiotic stress conditions (Figure 2), while, at the same time, allowing for increased drawdown of atmospheric CO2 to stabilized carbon pools in the soil.

Acknowledgments

Included in Title Page due to: Blinded manuscript (no author details): The main body of the paper (including the references, figures, tables and any acknowledgements) should not include any identifying information, such as the authors' names or affiliations. Acknowledgments

Funding was provided by the Pacific Northwest National Laboratory (PNNL) Laboratory-Directed Research and Development (LDRD) Initiative integrated Plant-Atmosphere-Soil System (iPASS; PNNL Project # 204412), and in part by US Department of Energy (DOE) Contract DE-AC05-76RL01830 with PNNL.

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Table 1. Molecular studies on alleviating abiotic stresses in plants using PGPRs.

Bacterial Species Plant Species Methodology Molecular Mechanism Identified Reference

P. fluorescens FPT9601-T5 Arabidopsis Microarray analysis Identification of 200 genes as differently expressed in bacterial-treated plants (Wang et al., 2005)

Gluconacetobacter diazotrophicus PAL5 Sugar cane Illumina sequencing (HiSeq 2000 system) Activation the ABA-dependent signaling genes conferring drought tolerance (Vargas et al., 2014)

Bacillus megaterium BP17 Arabidopsis Microarray based gene expression analysis Identification of 150 genes differentially expressed in bacterial treated plants (Vibhuti et al., 2017)

Streptomyces spp. viz., S. diastaticus, S. fradiae, S. olivochromogenes, S. collinus, S. ossamyceticus, and S. griseus Chickpea Gene expression profiling Increase in defense related enzymes in inoculated plants (Singh and Gaur, 2017)

Burkholderia phytofirmans PsJN Arabidopsis Quantitative gene expression profiling Auxin and ethylene hormone homeostasis in plant growth promotion (Poupin et al., 2013)

Pseudomonas putida MTCC5279 Arabidopsis leaves Microarray analysis Over expression of 520 genes and repression of 364 genes; up regulated (Srivastava et al., 2012)

genes included growth hormone and amino acid syntheses, ABA signaling and ethylene suppression, Ca2+ dependent signaling

Azospirillum brasilense Sp245 Rice qRT-PCR Increase in nitrogen fixation and an increase in the expression of ethylene receptors (Vargas et al., 2012)

Dietzia natronolimnaea STR1 Wheat qRT-PCR Confers salinity tolerance by modulating the transcriptional of salinity tolerance genes (Bharti et al., 2016)

Bacillus licheniformis K11 Pepper PCR and 2D PAGE Confers drought resistance by differential regulation of stress proteins and auxin and ACC deaminase production (Lim and Kim, 2013)

Pseudomonas simiae strain AU Soy bean MALDI-MS/MS and western blotting Facilitates induced systemic tolerance in soybean seedlings by producing a volatile organic blend (Vaishnav et al., 2015)

Bacillus subtilis GB03 Arabidopsis RT-PCR Confers salt tolerance by regulating the Na+ transporter in by up and down regulation of HKT1 in shoots and roots (Zhang et al., 2010)

Burkholdera cepacia SE4, Promicromonospora sp. SE188 and Acinetobacter calcoaceticus SE370 Cucumber Gas chromatography and enzyme activity assays Confers drought and salinity tolerance in inoculated plants by reducing catalase, peroxidase; and increasing salicylic acid and gibberellin (Kang et al., 2014a)

Enterobacter sp. UPMR18 Okra RT-PCR Salt tolerance by increasing antioxidant activity and up regulation of ROS pathway genes (Habib et al., 2016)

Pseudomonas fluorescens strain Arabidopsis Microarray analysis and Results in differential up regulation of (van de Mortel et

SS101 untargeted approximately 1,910 al., 2012)

metabolomics genes and 50

using LC-QTOF- metabolites in treated

MS plants appose to

untreated plants

Figure Legends:

Figure 1. Interactions between the rhizosphere and other components of the plant ecosystem. Carbon (C) enters the soil as root exudates or via decomposition of root or aboveground biomass. In the soil, C exists in root or microbial biomass, as bioavailable labile organic C, or as more recalcitrant C. Carbon exits the soil as direct emissions, or via root or microbial respiration, with microbial-mediated soil respiration being the major source of CO2 from terrestrial ecosystems. Carbon is also lost from the ecosystem as volatile organic compounds (VOCs) and methane (CH4).

Figure 2. Utilization of symbiotic root-microbe relationships, advanced synthetic biology tools and/or the combination of both approaches can be used to alleviate abiotic stress conditions in the rhizosphere. (1) PGPR provide the plant with a compound that is synthesized by the bacterium, for example phytohormones, facilitate the uptake of certain nutrients from the environment, or can act through various mechanisms as biocontrol agents. (2) Dissecting plant-PGPR interactions at the molecular level reveals valuable information that can be used to engineer (3) plants and/or (4) bacteria for the purpose of rhizosphere engineering and improving plant productivity under harsh environmental conditions.

Beneficial root-microbe interaction

Phyto hormone production

Plant Growth Promoting Bacteria (PGPBs)

Increasing Root Biomass Production and Whole Plant Productivity

Nutrient uptake

Synthetic biology

Dissection of Symbiotic Plant-Microbe Interactions

Biocontrol

Figure 2. Utilization of symbiotic root-microbe relationships, advanced synthetic biology tools and/or the combination of both approaches can be used to alleviate abiotic stress conditions in the rhizosphere. (1) PGPR provide the plant with a compound that is synthesized by the bacterium, for example phytohormones, facilitate the uptake of certain nutrients from the environment, or can act through various mechanisms as biocontrol agents. (2) Dissecting plant-PGPR interactions at the molecular level reveals valuable information that can be used to engineer (3) plants and/or (4) bacteria for the purpose of rhizosphere engineering and improving plant productivity under harsh environmental conditions.