Chapter 12

Root Secretions: Interrelating Genesand Molecules to MicrobialAssociations. Is It All That Simple?

Meredith L. Biedrzycki and Harsh P. BaisPlant and Soil Sciences Department,USA; Delware Biotechnology Institute,USA

12.1 PLANT ROOTCOMMUNICATION: THE BASICS

In order to survive a sessile lifestyle in an ever-changingenvironment, plants have evolved many mechanisms tosense and interact with the biotic and abiotic factors intheir surroundings. Plants are able to respond accordinglyto temperature, humidity, light, and soil hydrationchanges. Plants also respond to positive or negative inter-actions with other plants as well as interactions with her-bivores, pathogens, and symbionts. Interactions betweenplants and other biotic factors in their environment areoften facilitated through chemical signaling. These chem-ical signals can be released from the leaves as volatilesor secreted through the roots into the surrounding soil.Many studies have investigated the role of plant volatiles;however, less attention has been given to understandingchemical communication in roots until recent years.

Plants are supplied with nutrients, water, andanchorage from their industrious and plastic root systems(Malamy and Ryan, 2001). In addition to these veryimportant processes, roots also influence the surroundingsoil referred to as the rhizosphere (O’Connell et al., 1996;Hiltner, 1904; Bowen and Rovia, 1991). The rhizospherecan be described as having three main zones. The firstzone, referred to as the endorhizosphere, includes the rootcortical and endodermal tissues. The second zone, therhizoplane, encompasses the root epidermis and associ-ated mucilage, and the last zone or ectorhizosphere refers

Molecular Microbial Ecology of the Rhizosphere, Volume 1, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

to the soil near the root (Badri and Vivanco, 2009; Lynch,1987). The rhizosphere is a dynamic environment subjectto changes in water content, pH, and mineral depositions.Plants communicate with other soil organisms viachemicals secreted into the rhizosphere. The secretion ofthese chemicals is dependent on the plant species as wellas the physiological state and age of the plant. Roots cansecrete compounds in an active manner for a specificinteraction or in a constitutive manner for general signalsto other organisms. Root secretions can include secondarymetabolites such as phenols and flavonoids in additionto other sugars, organic acids, amino acids, and proteins(Bais et al., 2006; see Chapter 22). Production andsecretion of secondary metabolites as well as the othercompounds diverts energy from growth and reproductionof the plant. It has been shown that seedlings can secreteup to 30–40% of their photosynthetically fixed carboninto the rhizosphere, suggesting the importance of theseinteractions to plant survival (Badri and Vivanco, 2009;Whipps, 1990). To date, two potential mechanisms forsecretion of root compounds have been suggested. Thefirst is that secretions are released from root border cellsduring root growth and the second is that root secretionsare conveyed across the cell membrane directly into therhizosphere through diffusion or active transport (Baiset al., 2006 Vicre et al., 2005; Hentzer et al., 2003; Badriand Vivanco, 2009).

As mentioned previously, plants interact with diverselife forms in the soil and these interactions can be positive,

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138 Chapter 12 Root Secretions: Interrelating Genes and Molecules to Microbial Associations

negative, or neutral in nature. For the ease of scientificinvestigation, most plant interactions are studied in onlytwo organism systems. However, in nature it appears thatthere are many layers of communication present, resultingin tritrophic and multitrophic interactions between variouscombinations of plants, microbes, and herbivores. There-fore, it is important to touch upon these other interactionsto understand how they may influence the overall rhizo-spheric communities.

Allelopathy, one of the best known plant–plant inter-actions, describes a situation where one plant will secretea chemical into the soil to prevent the establishmentand growth of another plant or kill the same. Classicexamples of allelopathic plants that secrete chemicalsto gain advantage over other plants in the surroundingrhizosphere include rice, wheat, black walnut, Russianknapweed, and spotted knapweed (Bais et al., 2006). In aninteresting study, Centaurea maculosa, an invasive weed,has been found to secrete the allelochemical (±)-catechininto the surrounding rhizosphere to regulate populationand competition within the species. This regulation isaccomplished because seeds of C. maculosa recognizeconcentrations of the (±)-catechin in the rhizosphere andpostpone germination (Perry et al., 2005). In additionto moderating negative plant–plant interactions, rootsecretions are also known to be involved in beneficialplant–plant interactions. One well-known example ofthese favorable communications is in Phaseolus lunatus(lima bean) defense against spider mites. These plantssecrete compounds in the soil, which induce undamagedplants to release volatiles that attract predatory mites tocontrol the spider mite herbivory (Bais et al., 2006; Guer-rieri et al., 2002). Studies such as the above-mentioned C.maculosa system demonstrate that plants have the abilityto recognize members of their own species while morerecent studies in Cakile edentula and Arabidopsis thalianahave shown that plants can recognize not only thoseof their own species but also those that are geneticallyrelated versus nonrelated within the species (Dudleyand File, 2007; Biedrzycki et al., 2010). Although thesestudies suggest that this intraspecies recognition conferssome evolutionary benefit, these investigations mostimportantly shed light on the specificity of some plantinteractions in regard to recognition of secretions.

12.2 PLANT–MICROBEINTERACTIONS

In addition to plant–plant interactions, plant–microbeinteractions in the rhizosphere are ubiquitous. Mycor-rhizae or beneficial plant-associated fungi can be dividedinto two types, ectomycorrhizae and endomycorrhizae(also referred to as arbuscular mycorrhizae). Over 80% of

terrestrial plants have been found to associate with arbus-cular mycorrhizal fungi (AMF). In this interaction, plantsprovide the needed carbohydrates to the fungi, while thefungi provide limiting nutrients, most often phosphorus,to the plant. To initiate this symbiosis, mycorrhizae,triggered by root secretions such as amino acids, sugars,or secondary metabolites, particularly flavonoids, invadeplants roots and form branching arbuscules (Badri et al.,2009a; see Chapters 43, 51). The compounds secreted bythe plant roots often attract or support only specific fungalspecies. In Lotus japonicus, a plant-secreted strigolactonecompound was found to trigger hyphal branching in thefungi, allowing for more surface contact and exchangebetween the two organism (Bais et al., 2006; Akiyamaet al., 2005; Badri et al., 2009a; see Chapters 33, 34, 35).As mentioned, in the rhizosphere, arbuscular mycorrhizal(AM) fungal secretions have been shown to influence thecomposition of the surrounding bacterial communities byencouraging bacterial colonization leading to multitrophicinteractions (Badri et al., 2009a; Tolijander et al., 2007).

Similar to the aerial portion of the plant, roots aresusceptible to pathogenic microbial attack. Antimicrobialsecondary metabolites are produced by most plant speciesfor defense. These defense compounds can be secretedconstitutively, in order to deter or prevent pathogen attack,and are known as phytoanticipans. Compounds secreted inresponse to a pathogen attack are referred to as phytoalex-ins. One example of phytoalexin secretion is exhibited insweet basil, which secretes rosmarinic acid when chal-lenged with fungal cell wall extracts (Bais et al., 2002).

In addition to production and secretion of antimi-crobial compounds, plants have evolved intricaterelationships with soil bacteria to protect their healthand improve growth. These bacteria are often referredto as plant-growth-promoting rhizobacteria (PGPR) andinclude several genera of bacteria including Proteobacte-ria such as Pseudomonas, Burkholderia, and Firmicutes,including Bacillus (Badri et al., 2009a; Bais et al., 2006;Raaijmakers et al., 2009; see Chapter 53). Some PGPRshelp protect plant health by creating a “suppressive” soilby mechanisms such as niche exclusion and competitionfor nutrients with other microbial species as well as byproducing antifungal compounds. Additionally, thesePGPR may also trigger induced systemic resistance (ISR)in plants, which does not confer complete resistancebut does offer some protection by triggering jasmonateand ethylene pathways (Raaijmakers et al., 2009). Oneexample of this ISR induction was illustrated by Rudrappaet al. (2008) when they demonstrated that A. thalianaplants that were aerially infected with the plant pathogenPseudomonas syringae pv tomato (Pst DC3000) specifi-cally secreted l-malic acid and successfully recruited andpromoted the binding of the beneficial Bacillus subtilisFB17 to the roots. These results indicated a coordinated

12.3 Future Directions 139

response throughout the plant to induce root secretionsfor plant protection.

In addition to plant health benefits, PGPRs have alsobeen demonstrated to stimulate plant growth throughimprovement of nutrient acquisition such as by fixingatmospheric nitrogen (see Chapters 44, 45, 51, 52).Furthermore, PGPRs are known to directly secrete planthormones such as auxin, gibberellins, and cytokinins(Bais et al., 2006; Steenhoudt and Vanderleyden, 2000;see Chapters 27, 29). In order to establish these beneficialinteractions, the PGPRs most likely recognize secretedcues from the root such as carbohydrates and aminoacids, which serve as an energy source for the bacteria(Bais et al., 2006; Somers and Vanderleyden, 2004).Additionally, roots may secrete compounds that induceflagellar motility in the rhizospheric bacteria to promotebacterial swarming to the root (Bais et al., 2006; deWeert et al., 2002).

A specific group of microbes, referred to as endo-phytic bacteria, or bacteria that specifically colonize intothe root tissue (see Section 5) rather than being surfaceassociated can also be included in the PGPRs as theyalso are known to secrete plant growth hormones. Theseendophytic bacteria can be promiscuous in associationsor may have very specific interactions with species andeven cultivars of plants (Badri et al., 2009b; Ulrich et al.,2008; Taghavi et al., 2004). Another type of interactionthat is often very specific is that of rhizobia and legumesymbioses. In these symbioses, legume plants secreteflavonoid compounds into the surrounding rhizosphereunder limiting nitrogen conditions to attract rhizobia.Once the rhizobia recognize the specific flavonoids,NodD protein triggers transcription of nod genes, whichin turn produce the bacterial Nod factor. Legume rootsthen recognize this Nod factor and allow the rhizobia toinfect the root hairs and form the nitrogen-fixing nodules(Badri et al., 2009a; see Chapter 45). This symbiosis isintegral to many farming practices and is discussed indetail in later chapters (see Section 6).

Plant–microbe symbioses such as previously men-tioned have direct implications on improving plant growthand plant health; however, plants have also been foundto utilize rhizosphere bacteria for a more sinister pur-pose. In recent years, investigation regarding the speciesPhragmites australis has shown that this extremely inva-sive species utilizes root secretions for its toxicity againstnative species. Studies suggest that P. australis secretesthe phytotoxin gallic acid to kill surrounding competi-tors. Invasive P. australis rhizospheres were also foundto contain higher levels of bacteria that utilize gallotan-nin to release gallic acid, further increasing the toxicity.These rhizobacteria associations appear to be a contribut-ing factor in the allelopathic nature of P. australis anddemonstrate how plant-specific secretions can modify the

rhizosphere composition to aid the plant in invasion (Bainset al., 2009; Rudrappa et al., 2008).

As mentioned earlier, composition of root secretionsmay depend on the plant species and physiological stateof the plant as demonstrated when root secretions of rice,barley, and wheat have been collected and were deter-mined to have a different composition despite uniformgrowth conditions (Fan et al., 2001). Micallef et al. (2009;see Chapter 24) have made progress in determining thatspecific genetic backgrounds even within a species caninfluence the composition of root secretions. A highperformance liquid chromatography (HPLC) analysisof root secretions from eight different Arabidopsisaccessions revealed differences in secretion patternsbetween the ecotypes. Further analysis of plant–microbeinteractions revealed that the individual accessions haddifferences in the presence and abundance of bacterialcommunities (Micallef et al. 2009). This clearly illustratesthat plants, whether passively or actively, have the abilityto influence their rhizosphere, and it remains to be seenwhether rhizosphere microbes have this same ability toinfluence plant community growth.

12.3 FUTURE DIRECTIONS

Much of this chapter has illustrated how plants utilizesecretions to attract and interact with other plants andsoil microbes. Unfortunately, information regarding howthese secretion interactions are controlled and regulated isscarce. In a pioneering study, Loyola-Vargas et al. (2007)found, through addition of transport inhibitors, that ABCtransporters are involved in root secretions in Arabidopsis.ABC transporters have been identified in plants as wellas in bacteria, fungi, and animals and are often involvedin detoxification of xenobiotics. In plants, a subsequentexperiment showed that Arabidopsis ABC transportermutant root secretion profiles differed and that one ABCtransporter mutant significantly altered the soil microbecommunities in comparison to the wild-type plants (Badriet al., 2009b). In a related study, Biedrzycki et al. (2011)found that secretions involved in plant–plant recognitionwere also controlled by several ABC transports. Althoughthese studies point to some of the genes directly involvedwith the secretion process, genes involved in plants’ability to sense other plant and microbial compounds aswell as the abiotic environment still largely remain tobe identified. Additionally, genes involved downstreamof sensing and prior to secretion as well as many of thecompounds secreted continue to be a mystery. Furtherresearch in this area may allow for engineering of plantsbetter suited to their environments. Oger et al. (2004;see Chapter 110) have already demonstrated the abilityto engineer Lotus plants to secrete specific opines to

140 Chapter 12 Root Secretions: Interrelating Genes and Molecules to Microbial Associations

enrich rhizobacteria communities. A more completeunderstanding of the sensing, signaling, and secretion inrhizosphere plant–microbe communications may lead toimprovements in agriculture such as enhanced nutrientacquisition and improved plant health. This knowledgemay also be used to improve eradication of invasivespecies as well as for phytoremediation of toxic soilcontaminants (O’Connell et al., 1996; see Section 12).Therefore, continued investigation into root secretions,their genes, molecules, and microbial associations will benecessary to understand and apply these technologies.

ACKNOWLEDGMENT

H.P.B. acknowledges the support from NSF Award0923806.

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