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Year: 2018

Feed Your Friends: Do Plant Exudates Shape the Root Microbiome?
Sasse, Joelle ; Martinoia, Enrico ; Northen, Trent

DOI: https://doi.org/10.1016/j.tplants.2017.09.003

Posted at the Zurich Open Repository and Archive, University of Zurich
ZORA URL: https://doi.org/10.5167/uzh-148899
Journal Article
Accepted Version
Originally published at:
Sasse, Joelle; Martinoia, Enrico; Northen, Trent (2018). Feed Your Friends: Do Plant Exudates Shape the RootMicrobiome? Trends in Plant Science, 23(1):25-41.
DOI: https://doi.org/10.1016/j.tplants.2017.09.003

1Feed your friends: do plant exudates shape the root2

microbiome?

3
4
5

Joelle Sasse1, Enrico Martinoia2, Trent Northen1,3*

6
7

1Lawrence Berkeley National Laboratory, Berkeley, USA, 94720

82Department

9

Switzerland, 8008

10of Plant and Microbial Biology, University of Zurich, Zurich,

3 Joint Genome Institute, Walnut Creek, USA, 94958

11
12*Corresponding author:

13

trnorthen@lbl.gov (T. Northen)

14

www.northenlab.org

15@Twitter: @northenlab

16
17

Keywords

18

Rhizosphere, microbiome, root exudates, transporter, metabolite networks,

19

symbiosis

20
21

22

Abstract

23

Plant health in natural environments depends on interactions with complex and24

dynamic communities comprised of macro- and microorganisms. While many

25

studies have provided insights into the composition of rhizosphere microbiomes

26(rhizobiomes), little is known about if plants shape their rhizobiomes. We discuss

27

physiological factors of plants that may govern plant-microbe interactions, focusing

28

on root physiology and the role of root exudates.. As only few plant transport

29

proteins are known to be involved in root metabolite export, we suggest novel

30

families putatively involved in this process. Finally, building off of the features

31

discussed in this review, and in analogy to well-known symbiosis, we elaborate on a32

possible sequence of events governing rhizobiome assembly.

33

34

The root microbiome (rhizobiome)

35

Plant growth and yield in natural environments depend on a plethora of interactions

36

with bacteria and fungi [1] (one example is discussed in Box 1). The microbial

37

community associated with roots was proposed to be assembled in two steps: first,

38

the rhizosphere (see Glossary) is colonized by a subset of the bulk soil community

39

and second, the rhizoplane and the endosphere are colonized by a subset of the40

rhizosphere community [2][3]. Intriguingly, a set of recurring plant-associated41

microbes has emerged (core microbiome), [2,4]. This review is focused on how

42plants shape their rhizobiome: On the one hand, common factors among plants

43

likely lead to the assembly of the core microbiome. On the other hand, factors

44

specific to certain plants result in the association microbes that are not members of45

the core microbiome. Here, we discuss evidence that plant genetic factors,

46

specifically root morphology and root exudation, shape rhizobiomes.

47

Initial evidence for an influence of plant genotype on rhizobiome composition was48

that similar rhizobiomes assembled in association with arabidopsis (Arabidopsis

49

thaliana) and barley (Hordeum vulgare) grown in the same experimental conditions,50

however, displaying different relative abundances and some specific taxonomic

51

groups [5]. A correlation between phylogenetic host distance and rhizobiome

52

clustering was described for Poaceae species [6], for distant relatives of arabidopsis

53[7], for rice varieties [3], and for maize lines (Zea mays) [6], but not for closely54

related Arabidopsis species and ecotypes [7]. Distinct rhizobiomes were also

55

described for domesticated plants, such as barley, maize, agave (Agave sp.), beet

56

(Beta vulgaris), and lettuce (Lactuca sp.), compared to their respective wild relatives

57[5,8-11]. Interestingly, not all plants have a rhizobiome distinct from bulk soil: Some58

species such as maize and lotus (Lotus japonicus) [12-15] assembled a distinct

59

rhizobiome, whereas other species such as arabidopsis and rice assembled a60

rhizobiome similar to bulk soil [3,5,7]. The former species display a strong, and the61latter a weak rhizosphere effect (see Key Figure 1). The cause of this phenomenon

62

is currently unknown. The strength of the rhizosphere effect varies with the plant’s

63developmental stage [16-18]. Similarly, root exudation [16], and microbial

64

communities were found to change with the plant’s age.

65

Furthermore, distinct rhizobiomes were associated with different developmental

66

stages of arabidopsis [16,19], rice [3], and Avena fatua grown during two

67

consecutive seasons [20]. Pioneering studies demonstrated the ability of microbes

68

to alter plant development [22]. Overall, it seems evident that host genotype,69

domestication, and plant development influence the composition of rhizobiomes.

70

Alternatively to plant developmental stage, residence time of plants in soil was

71

discussed as a hypothesis for successive microbiomes [21]. These contrasting72

results might be partially explained by differing environmental influences, host73

plants, or soils, and additional work is needed to resolve these questions.

74

In this review, we discuss root morphology and root exudates as two genetic factors

75

shaping plant-microbiome interactions, and we examine the following aspects: (i)76

how root morphology and border cells affect rhizobiomes, (ii) how plant exudates

77

shape the rhizobiome, and (iii) possible plant transport proteins involved in78

exudation. Key Figure 1 displays a general overview on exometabolite networks in79

the rhizosphere, and Box 1 illustrates the interplay between root exudates, border80

cells, and rhizobiomes in phytoremediation. In the last chapter of this review, we

81

integrate these ideas into a possible scenario of rhizobiome assembly.

82
83
84
85
86
87
88

Root physiological features shape rhizobiomes and exudation

89

Rhizobiomes are influenced by their spatial orientation towards roots in two ways:

90

First, the radial proximity of microbial communities to roots defines community

91

complexity and composition, as described in many recent publications [3,19,23],92

and as outlined by the two-step model of microbial root colonization mentioned

93

above [2]. Second, the lateral position of microbes along a root shapes the94

community, as exemplified by early studies {DeAngelis:2008da}{DeAngelis:2005ea}.

95

Importantly, recent microbiome studies take into consideration the former, but not96

the latter aspect. In this section, we discuss specific microbial associations with97

various root regions, and the role of spatially distinct root exudation.

98

Root tips are the first tissues that make contact with bulk soil: root tips are99

associated with the highest numbers of active bacteria compared to other root100

tissues, and likely select microbes in an active manner [24]. The root elongation

101

zone was specifically colonized by Bacillus subtilis, which suggests a particular role102of this zone in plant-microbe interactions [25]. Mature root zones featured a103

microbial community distinct from root tips [25]. Their community included

104

decomposers [11,26], which could be involved in degradation of dead cells shedding105

from old root parts [27]. Similarly, lateral roots were associated with distinct

106

microbial communities, differing between tips and bases, as well as between

107

different types of lateral roots [14].

108

One trait influencing the differential microbial colonization of root tissues could be109

the differential exudation profiles of the distinct root parts. This is illustrated in the110

following example: Cluster roots are densely packed lateral roots formed by some111

plants growing on extremely nutrient-poor soils, that exude high amounts of organic

112

acids, and in some cases protons, to solubilize phosphate [28]. The low pH and113

carboxylate-rich rhizosphere of cluster roots is associated with a specialized114

rhizobiome, dominated by Burkholderia species that metabolize citrate and oxalate

115[29]. Besides organic acids, mature cluster roots also exude isoflavonoids and fungal

116

cell wall degrading enzymes, leading to a decrease in bacterial abundance, as well as

117

fungal sporulation [30]. Taken together, cluster root exudates not only solubilize

118

phosphate, but also regulate microbes in a way that they do not interfere with

119

phosphate uptake. Beyond this example, spatial patterns of metabolite exudation120

are largely unexplored. We hypothesize that such patterns exist in all root systems

121

for the following reasons: (i) Spatially distinct organic acid exudation is a trait of all

122root systems (Table 1, Box 1). (ii) Spatially distinct exudation was similarly detected123

for strigolactones, amino acids, and sugars (Table 1) [31,32]. (iii) Root nutrient

124

uptake, which is sometimes coupled to proton transport, can also exhibit spatial

125

patterns (Table 1). Overall, spatially defined metabolite exudation by distinct root

126

parts is likely an important factor in structuring the rhizobiome. Future studies

127

should aim at characterizing spatially distinct rhizobiomes and their functional

128

traits, and at investigating spatially distinct root exudation.

129
130

Root border cells and mucilage shape plant – microbe interactions

131

Root tips are not only associated with high numbers of bacteria ([26], see above),132

they also produce border cells and mucilage (Key Figure 1), crucial for plant-

133

microbe interactions. Depending on the root meristem organization, border cells are

134

released into the rhizosphere either as single cells or as border-like cells (these cells

135

remain attached to each other). Residence time in the soil is different for the two

136

types of border cells: Single maize border cells stayed alive in soil for months, likely

137

due to the presence of starch deposits [33], whereas arabidopsis border-like cells

138

survived only two weeks [34]. Border cells have a transcriptional profile distinct

139

from root tip wells, with overall lower primary and higher secondary metabolism

140[33]. ABC transporters constituted a large fraction of differentially expressed genes,

141

which is consistent with transport of secondary metabolites [33,35]. Secondary142

metabolites likely are central to the role of border cells in defense against pathogens

143[36-38].

144

Pathogen attack cannot only result in higher border cell production and release [36-14538], but also in higher mucilage production by border cells and root tip cells:

146

Mucilage contains proteins with antimicrobial functions [36,39,40], as well as

147

extracellular DNA involved in defense against fungi [41] and certain bacteria [42].

148

Importantly, mucilage is also produced in nonpathogenic conditions, as it serves as

149

lubricant for the root environment and stabilizes soil particles [43]. Interestingly,

150

mucilage also provides distinct carbon sources for microbes, thus influencing

151

rhizobiome composition [44,45].

152

Border cells similarly interact with nonpathogenic microbes (Figure 2): they release153

flavonoids that attract rhizobia, uncharacterized compsounds that induce branching154

of mycorrhizal hyphae, and arabinogalactans that trigger biofilm formation of155

specific beneficial bacteria (Box 2) [33,34,46,47]. The full extent of how border cells

156

and mucilage shape root – microbe interactions remains unclear. It is tempting to

157

speculate that the border cells’ specialized metabolism results in a distinct

158

exudation profile of not only proteins and mucilage, but also low-molecular weight

159

compounds that could serve as microbial nutrients, or as signaling compounds.

160

Further research could focus on the genetic and physiological differences between

161

border cells and border-like cells, as well as on the transport proteins involved in

162

exudation of low-molecular compounds, DNA, and proteins.

163
164

How microbial communities interact, and the influence of plant exudates

165

Plant-microbe interactions are not only defined by plant root morphology and plant-

166

derived exudates, but also by microbe-microbe interactions (Key Figure 1). Thus, in167

this section, we focus further on microbial communities. Specifically, we discuss (i)168

how plant exudates influence microbial diversity, (ii) how plant-responsive

169

microbes are identified, (iii) how microbes interact, and (iv) how mycorrhizal fungi

170

influence root-bacteria interactions.

171

The rhizosphere serves as carbon-rich niche for the establishment of microbial

172

communities, in contrast to bulk soil, which is rapidly depleted from carbon and173

other nutrients by heterotrophic microbes. As the ability of microbes to metabolize

174

plant-derived exometabolites might determine their success in microbial

175

community, several studies investigated whether the diversity of plant exudates

176

correlated with microbial diversity. Some studies indeed found higher plant177

diversity associated with higher microbial diversity [48,49], and the addition of a178

diverse exudate mix to plant monocultures increased microbial diversity [50].

179Interestingly, isolates from soils with a diverse plant community consistently

180

exhibited less narrow niches and displayed less resource competition than isolates

181

from low plant diversity environments [51,52]. Although on a global scale,182

environmental factors had a larger impact on microbial diversity than plant183

diversity [49], we conclude that on a local scale, high plant diversity likely promotes

184

a diverse microbial community.

185

The large diversity of microbial communities is a current challenge for plant-

186

microbe research, as it is impractical to study questions such as how members of a187

community interact, and what specific traits a microbial community possesses.

188

Therefore, many studies currently aim at identifying the subset of microbes

189

responsive to plants. Strikingly, only 7% of bulk soil microbes increased in

190

abundance in the rhizosphere compared to buk soil [26], which lowers the number

191

of taxa to investigate from thousands to hundreds. Other approaches in identifying

192

plant responsive microbes focused on transcriptional profiling: Compared to soil-

193

abundant microbes, plant-associated microbes exhibited distinct transcriptional

194

responses to plant exudates [53,54], and intriguingly, displayed distinct

195

phylogenetic clustering [18,53]. Network analyses further revealed that rhizosphere196

microbes displayed higher levels of interactions than bulk soil microbes [55]. These

197

studies illustrate the potential for the identification of a distinct set of plant-

198

responsive microbes.

199

The above points illustrate how plants influence microbial communities. But

200

evidently, the members of microbial communities as well interact with each other:

201

Compellingly, it is still unclear if microbe-microbe interactions are predominantly202

positive or negative. Network analyses reported predominantly positive intra-

203

kingdom interactions [55,56]. In contrast, laboratory growth assays identified

204

competition as the major factor in shaping isolate communities, and cooperation

205

could only be detected for 6-10% of the isolates [57,58]. One major difference

206

between the two experimental approaches is that the former investigates a natural

207

system, whereas the latter is based on the ability to culture microbes. Isolation of208

microbes introduces a bias, since it can select against cooperators precluding

209

obligate syntrophs. Further evidence that at least some microbes avoid competition210

was provided by co-cultivation experiments: Environmental isolates (i) displayed

211high substrate specialization [60], (ii) did not necessarily take up the compound212

with the highest energy [61], and (iii) diverged in substrate use when cultivated for213

several generations [51,58]. In addition, some metabolites exuded by microbes214

could be metabolized by others [60], suggesting a potential for cross-feeding

215

between community members. The above findings suggest complex interactions of216

microbes. It remains to be resolved in which situation competition or cooperation

217

dominates communities. It is evident however, that microbial interactions are based

218

on altered gene expression. Microbes responded to competing bacteria [62] or even219

close relatives [63] by differentially regulating genes involved in metabolite

220

exudation and transport processes [62,64], making the study of microbial

221

transporters a compelling topic for future studies. Metabolite uptake, release, and222

sensing are clearly important factors in shaping microbial communities.

223

Metabolite turnover in soil is influenced by plants, but also by functionally diverse

224

bacteria, fungi and animals [65]. Plant-fungal and plant-animal interactions in the225

rhizosphere go beyond the scope of this review, and are discussed elsewhere [65-22667]. Here, we provide a few brief examples focusing on the impacts of mycorrhiza on

227

rhizobiomes and exometabolite turnover. Endomycorrhizal fungi receive a

228

significant fraction of fixed carbon by plants (Box 2). Interestingly, these fungi also229

exude sugars [68], shaping a distinct bacterial community [68,69]. Likewise,

230

ectomycorrhiza receive carbon from plants, and feature a dynamic bacterial

231

community [70]; they even participate in plant-to-plant carbon transport [71]. The

232

field of fungal microbiomes is nascent: If and how fungi control exudation, whether

233

fungal microbiomes have beneficial functions, and how plant and fungal

234

microbiomes influence each other are unknowns. Although many open questions

235

remain, these recent findings already suggest that a holistic view of rhizosphere236

nutrient cycling and signaling exchange via exometabolites requires a whole237

community approach including all domains of life.

238

239

Exudates are diverse and dynamic

240

Plant exudates shape microbial communities. Overall, plants exude up to 20% of

241

fixed carbon and 15% of nitrogen [66,72], which includes an array of simple

242

molecules such as sugars, organic acids, and secondary metabolites, as well as

243

complex polymers such as mucilage (see Table 1, Figure 1, Figure 2). Although every

244

plant produces exudates, the amount and composition of root exudates varies. First,

245

exudation is defined by the hosts genotype, as observed in the distinct exudation

246

patterns of 19 arabidopsis accessions [73]: Strikingly, the amount of variation

247

between the accessions depended on the metabolite class investigated.

248

Glucosinolates displayed most, flavonoids medium, and phenylpropanoids low

249

variability [74]. Second, exudation changed with plant developmental stage: With

250

increasing age, arabidopsis sugar exudation decreased, and amino acid and phenolic

251

exudation increased [16]. Third, exudation is modulated by abiotic stresses: The

252

amounts of exuded amino acids, sugars, and organic acids changed in maize grown253

in phosphate, iron, nitrogen, or potassium deficient conditions [54]. In addition,254

phosphate deficient arabidopsis plants increased coumarin and oligolignol

255

exudation [75], heavy metals treated poplar (Populus tremula) induced organic acid256

exudation [76], and zinc deficient wheat increased phytosiderophore exudation

257[77]. Differential exudation is a plausible mechanism by which plants could

258

modulate their interaction with microbes, as exemplified by the correlation between

259

exudation patterns and rhizobiome variation reported for eight arabidopsis

260

accessions [78]. Differential exudation modulated by transport proteins is discussed

261

in the next paragraph.

262

Characterized and putative plant transporters for exudation

263

Plant-derived exometabolites need to cross at least one membrane to transit from264

the cytoplasm of root cells into the rhizosphere. There is considerable discussion as265

to which degree plants are able to regulate this transport. In general, different

266

modes of transport could be envisioned: First, small, hydrophilic compounds could

267

diffuse from the root into the rhizosphere, driven by the large concentration

268

gradient [27,79]. Second, channel proteins could facilitate such diffusion. Third,

269

active (ATP-driven) or secondary active (proton gradient driven) transporters could

270

shuttle compounds across membranes against a concentration gradient. Diffusion of271

compounds can only be relevant in young root tissue, which is still devoid of272

Casparian strips or suberized endodermis that both block apoplasmic flow in273

adult tissues. Transport proteins involved in exudation are mostly elusive. From a274

conceptual point of view, plasma membrane localized exporters likely have a direct,275

and vacuolar transporters an indirect effect on exudation. The vacuole is a major

276

storage organelle for many metabolites detected in exudates, such as sugars, organic

277

acids, and secondary metabolites [80]. Alteration of vacuolar transporter levels

278

impacts vacuolar and cytosolic concentrations, and thus, can influence metabolite

279

exudation into the rhizosphere.

280

The few characterized transporters involved in exudation are essential for transport

281

of specific compounds (Figure 2, Box 2) [32,81], and are presented in Table 1. Since282

only few transporters involved in exudation have been characterized, we suggest

283

additional families that might be involved in the process. To complete the picture of284

metabolite exchange between roots and soil, Table 1 additionally contains a few

285

important plasma membrane localized metabolite uptake systems. Below, we

286

discuss the evidence for transport processes involved in import and exudation of287

compounds detected in root exudates, such as sugars, organic acids, and secondary

288

metabolites

289

Sugars constitute a significant fraction of exudates, and are a main carbon source

290

for microbes [14,43]. Interestingly, many more sugar uptake than release systems

291

have been described. The STPs (Sugar Transport Protein) utilize high extracellular

292

proton levels to import sugars, and mutation of STPs lead to higher external sugar

293

levels [82,83]. SWEETs (Sugars Will Eventually Be Exported Transporters) are sugar

294

uniporters, and all root-expressed members localize to the vacuole [84,85]. Due to295

an alteration of the root sugar homeostasis, SWEET mutant plants exhibited higher

296

sugar export from roots than wild type plants, and were more susceptible to disease

297[84]. Intriguingly, no transporters directly exporting sugars into the rhizosphere298

have been characterized so far, and it is debated whether sugar exudation is a299transport-driven process at all [27]. Potential evidence for passive sugar efflux was

300

supported by the observation of higher sucrose concentrations around young,301

permeable root tissue than around older, less permeable root tissue [31]. However,302

as sugars are synthesized in leaves, sugars still need to be unloaded either from303

phloem or from root cells to be exuded into the rhizosphere, a process likely

304

depending on transporters due to the hydrophilic nature of sugars. A further

305

indication of presence of elusive transporters is the differential sugar exudation in

306

various environments, as was shown for example for maize in potassium,307

phosphate, or iron deficient conditions [86-89].

308

Sugar alcohols are imported by secondary active proteins with broad substrate309

specificity (Table 1), whereas modes of export are enigmatic. Sugar phosphates are310

involved in intracellular carbohydrate metabolism, and plastid-localized sugar -311

phosphate co-transporters were reported in several species [90]. Although sugar

312

phosphates are detected in exudates, neither import nor export mechanisms are313

currently characterized.

314

Amino acids are recognized by microbial chemoreceptors crucial for early steps of315

root colonization [91], making amino acids an important fraction of exudates.

316

Modulation of amino acid transport could either be a means of communication with317

microbes, or a response to microbial presence. Amino acid uptake is mediated by318

several transporter families with broad substrate specificity (Table 1, [92]). Amino

319acid exudation is affected by several transporters expressed in vascular tissue:

320

mutation of phloem-localized UmamiTs resulted in lower amino acid exudation [93],321

whereas mutation of xylem-localized GDUs (Glutamine dumper) caused increased

322

exudation [94]. Although no plasma membrane localized amino acid exporters are

323

characterized yet, several lines of evidence suggest their presence. First, higher

324

tryptophan exudation from older root zones than younger parts [31] suggests

325

involvement of transport proteins in exudation, due to the fully formed Casparian326

strips and thick cell walls in mature root parts interfering with diffusion. Second,327

concentration differences between amino acids in root exudates and root extracts328

are not the same for all the amino acids [92], suggesting the selective transport of at329

least some amino acids. Third, various transporter families exhibit bidirectional

330

amino acid transport characteristics in heterologous systems (Table 1), and could

331

potentially be involved in amino acid exudation.

332

Organic acids constitute a large fraction of exudates, and are microbial nutrients.

333

No importers are characterized so far, but the release of malate and citrate by ALMT

334

(Aluminium activated malate transporters) and MATE (multidrug and toxic

335

compound extrusion) families are among the few well-understood examples of336

transporters involved in exudation (Table 1, Figure 2). Activity of members of both337

families is often modulated by metal ions (Box 1) and by microbes (Box 2).

338

Uncharacterized ALMT and MATE family members are primary candidates for339

exporters of other organic acids due to their similarity to already characterized340

members, their plasma membrane localization, and their function as proton341

antiporters .

342

Nucleotides are imported by secondary active transporters, but exudation remains

343

elusive (Table 1) [95,96]. It is well established that extracellular ATP has a signaling344

function, and ABC transporters were proposed to mediate cellular export [97,98].

345

Peptide uptake was transporter-mediated in heterologous systems, and a role of346

ABC transporters in peptide exudation was suggested (Table 1).

347

Fatty acid transport is necessary for mycorrhizal symbiosis: mycorrhizal fungi

348

depended on their hosts for synthesis of certain fatty acids [99], and the current

349

model includes transport of lipids by ABCG proteins in the symbiotic membrane

350[99,100]. One ABCG member, STR, was shown to be required for mycorrhization351

previously [101]. Interestingly, arabidopsis ABCG transporters were similarly

352

shown to export fatty acids for cutin synthesis in aboveground tissues (Table 1).

353

Lipid transport was not only required for symbiotic interactions, but also for354

pathogen colonization [99]. Fatty acids are detected in root exudates (Table 1), but355

the mode of lipid exudation into the rhizosphere is still to be discovered. A role in356

lipid exudation could be envisioned for root-expressed ABCG members (Table 1,

357

Figure 2).

358

Secondary metabolites are ubiquitous in root exudates, and ABC transporters

359

likely candidates for specialized metabolite transport into the rhizosphere. A

360

distinct exudation profile was described for seven ABC mutants [102], and one

361

mutant line displayed an altered microbial community [103]. Although the causal362

metabolites could not be identified, the authors noted transport of the same

363

compound by various proteins, and possible broad substrate specificity for some364

transporters [102]. In a later study, exudates of arabidopsis ABCG37/PDR9 mutant

365

lines were found to be deficient of several phenylpropanoids [104](Figure 2D).

366

Arabidopsis PDR9 was previously characterized as auxin precursor transporter

367[105], which suggests a broad substrate specificity for PDR9. Interestingly, a PDR9368

homolog was highly expressed in cluster roots of white lupin devoid of phosphate369[106], illustrating PDR9 involvement in response to various abiotic stresses. These370

studies illustrate the potential for discovery of novel transporter functions in the371

ABC family, an excellent target for future studies investigating root exudation. In

372

addition, MATE proteins transport secondary metabolites into the vacuole, and373

plasma membrane localized members could be involved in secondary metabolite

374

exudation as well.

375

In summary, more transport proteins involved in metabolite import into roots than376

export from roots are reported (see Table 1). The characterization of additional

377

transport families involved in exudation will enable the generation of mutant lines

378

that are devoid in exudation of specific metabolites. Such lines could be used to379

investigate the correlation of exudation profiles and microbial communities.

380

How do rhizobiomes assemble?

381

Plant-derived transporters and exometabolites are intrinsic to plant-mycorrhizal

382

and -rhizobial symbioses (Box 2). We speculate that although there is paucity of383

evidence, plants analogously select for a beneficial rhizobiome: Plants evolved in the384

presence of microbes, a subset of which benefits plant growth. Thus, we hypothesize

385that over millennia, plant exudation via active transport processes evolved with386

substrate specificity of plant-associated bacteria. In Box 2, we discuss exudates and387

other steps involved in root microbiome assembly, analogously to establishment of388

plant- mycorrhizal and -rhizobial symbioses. However, intense future research is389

needed to reveal the precise mechanisms governing plant microbiomes assembly,390

and possible beneficial functions of the microbial community.

391

The major mechanisms by which plants are thought to modulate microbial

392

interactions currently include: (i) modulation of their exudate profiles (alteration of393

biosynthesis and/or transport of microbial substrates and signaling molecules), (ii)394root morphology (number and length of roots, root surface), and (iii) regulation of395

immune system activities (tolerance or avoidance). In turn, mechanisms for396

successful rhizosphere colonization by soil microbes requires that they (i) are397

metabolically active (catabolism of exudates), (ii) sense the plant (receptors for

398

exudates), (iii) move towards the root (chemotaxis, mobility), and (iv) successfully399

compete with other microbes for root niches (physical colonization, substrate

400

competition, defense against toxins). In addition to this, for a successful colonization

401of the rhizoplane or root tissue, microbes must e able to (v) attach to the surface

402(cell wall sensing, biofilm formation), or (vi) enter root tissue (evasion/

403

manipulation of immune system).

404

Despite apparent parallels between plant microbiomes and aforementioned

405

symbioses, plant microbiomes have some specific aspects: First, microbiomes are406

detected in all environmental conditions, whereas mycorrhizal and rhizobial

407

symbioses are induced in specific circumstances. Second, microbiomes are present

408

on various tissues, but rhizobia and mycorrhiza interface with roots only. Third,

409

microbiomes comprise of many members, whereas the aforementioned symbioses

410

persist between two predominant partners. Fourth, although most members of the411

microbiome originate from the environment [2,4,107] similar to rhizobia and412

mycorrhiza, there is evidence that some endophytes may be vertically transmitted413

via seeds [108-111]. Future research should focus on the factors involved in

414

microbiome assembly, the relative contribution of epi- and endophytes to415

microbiomes, and the signaling crosstalk between plants and microbial

416

communities.

417

Concluding remarks and future directions

418

Rhizobiome assembly and the plants involvement in this process are currently

419

enigmatic. Here, we discussed multiple factors shaping the rhizobiome, including420

host genotype and development, root morphology, border cells and mucilage, and421

root exudates. Root exudation is a dynamic process, likely dependent on a plethora

422

or transporters that are mostly uncharacterized. Spatially defined exudation likely

423

results in the distinct microbial communities that were observed to be associated424with specific root parts. The success of microbial colonization of the rhizosphere425

depends

426

competitiveness and cooperativeness. Further, endophytes likely form biofilms on427

the root surface, and cope with the plant immune system. Although some factors

428

shaping root microbiomes emerge, many open questions remain (see Outstanding

429

questions).

430

One major challenge will be to analyze root exudation in natural settings. Due to the431

chemical complexity of soil, exudation is traditionally analyzed in hydroponic

432

culture [14,16,73,89], an environment distant from more natural settings of plant433

microbiome studies. Further, novel technologies enabling high-throughput

434

screening of putative transporters against possible substrates are needed to reveal

435

the impact of the respective substrates on the rhizobiome, and, in turn, on plant436

health. An increased understanding of root morphology, exudation, and involved

437

transporters will likely enable the engineering or breeding of plants with altered438

abilities to interact with specific beneficial microbes or pathogens. This needs to be439

complemented with a greatly improved understanding of the substrate preferences440of plant-associated microbes, their interactions, and the mechanisms through which

441

they benefit the plant. A holistic understanding of the functions of a healthy plant442

rhizobiome would enable the directed design of customized microbial communities.

443

With this, specific plants in a given environment could be tailored to a specific444

purpose, such as phytoremediation, stress resistance, altered plant development, or

445

increased yield.

onseveralaspectssuchaschemotaxis,

substrate

specificity,

446

Acknowledgements

447

We thank Estelle Couradeau, Kateryna Zhalnina and Pascal Schläpfer for critical448

reading of the manuscript J.S. is supported by the National Science Foundation (NSF449

Proposal 1617020), and work in the laboratory of EM is supported by the Swiss450

National Foundation.

451

452

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Plant Cell 19, 2023–2038

822

Box1. Phytoremediation: interplay of exudates, border cells, and rhizobiomes

823

Plants grown on soils contaminated with heavy metals and organic pollutants824(phytoremediation) assemble a rhizobiome distinct from plants grown on non-825

contaminated rhizosphere, or bulk soil [112-114], which supports plant growth826[115,116] and higher heavy metal uptake [117]. Consequently, efforts have aimed at827

increasing the phytoremediation potential of heavy metal accumulators by

828

combining them with specific microbial communities. However, due to limited

829

understanding of the plant – microbe – environment interplay, these endeavors

830

have had limited success so far [112,113]. Below, we discuss both, the response of831

plants and microbes to contaminated soils.

832

Plants display distinct responses to contaminated soils: legumes exhibited a833

systemic response, and grasses a more local response [118]. Various wheat cultivars

834

displayed varying degrees of heavy metal tolerance, which were not only associated835

with distinct rhizobiomes [119], but also with the ability to exude organic acids [76].

836Tolerant lines increased expression of specific genes, such as the malate transporter

837

ALMT1 (Box 2, Table 1) and organic acid exporters of the MATE family [120].

838

Organic acid exudation has two main effects: First, (heavy) metal ions are chelated,839

and second, they can act as anion exchanger and release tightly bound phosphate,

840

supporting plant growth [112,113]. Increased organic acid exudation could also lead841

to increased nutrient availability for the microbial community, and have signaling

842

functions. A second physiological response to protect the root from heavy metals is843

an increased production and shedding of border cells accumulating heavy metals

844[121]. The interplay of border cell production and differential exudation by845

alteration of transporter abundance not only determines the plants performance on

846

contaminated soils, but also the environment for the microbial community.

847

Similar to plants, microbes in contaminated soils are under selective pressure from848

several sides: they have to tolerate the toxic environment, grow on root exudates,849

and compete for niches and resources [112,115]. It is thus no surprise that

850

rhizobiomes of contaminated soils were found to be generally less diverse

851

compared to other environments [115]. The addition of a substrate mix or the852

supply of plants with distinct exudation patterns could increase growth of853

specifically engineered or native microbes, leading to an increase of functional traits854

of the rhizobiome, and the phytoremediation potential of the microbial community.

855

In addition, a tritrophic bacteria-fungi-plant interactions on contaminated soil was

856

reported recently: microbes simultaneously increased arsenic tolerance in rice and857

resistance against disease [116]. Overall, further investigations of the roles of858

exudates, transporters, border cells, and bacterial and fungal communities will859

contribute to deciphering the effects of contaminated soils on plants, and lead to

860

more efficient phytoremediation procedures.

861

Box2. Is there a common theme of symbiosis?

862

The establishment of symbioses between plants and mycorrhiza or rhizobia is863

detailed in the literature, but assembly of plant-associated microbiomes remains

864

unclear. Here, we present a hypothesis on the assembly of a complex microbial

865

community in the rhizosphere that bases on the mechanism reported for the866

aforementioned symbioses (Figure I).

867

Plants induce symbioses with mycorrhiza and rhizobia in nutrient poor soils. The

868

symbionts are attracted by strigolactones exported by an ABCG type transporter

869

located in a specific root zone, or by flavonoids likely exported by a transporter of870

the same family (Figure 2A-B) [32,122,123]. Signaling molecules leading to the871

assembly of rhizobiomes are largely uncharacterized, but one example illustrates a872

symbiotic interaction with a beneficial microbe (Figure 2C): Pathogen-infected or873

elicitor treated arabidopsis plants increased ALMT1 expression and malic acid874

exudation (Table 1), which lead to specific attraction and root colonization of the875

biocontrol agent Bacillus subtilis, [81]. Interestingly, B. subtilis root colonization was

876

not malic acid dependent [81], suggesting presence of additional signaling

877

compounds.

878

Signaling compounds are similarly exuded by mycorrhiza and rhizobia:

879

Lipochitooligosaccharides (LCOSs) are required for the induction of symbiosis

880[124,125]. Mycorrhiza further require plant-derived cutin to attach to the root881

surface [126]. Some rhizosphere microbes produce N-acyl homoserine lactones

882(AHL) and volatile organic compounds (VOCs) that are sensed by plants [65].

883

Biofilm-derived exopolysaccharides similarly elicit plant transcriptional responses

884[127]. These compounds could be part of the plant – microbe crosstalk. Further,885

plant-derived cell wall polysaccharides and other signals [46] were shown to initiate

886

microbial root colonization and biofilm formation [23].

887

In a next step, plants respond to rhizobia and mycorrhiza by initiating the common

888

symbiosis pathway (SYM), altering gene expression and root morphology [128]. The

889

response of the immune system is distinct, with mycorrhiza eliciting and rhizobia

890

suppressing an initial pathogen response [129,130]. The immune system is also891

important in microbiome establishment (Figure 1): for example, the phytohormone

892

salicylic acid is not only involved in responses to microbial pathogens, but is also893

required for assembly of a typical microbiome [131]. Also, the genetic network for894

phosphate starvation signaling was found to influence the structure of microbiomes

895[132]. Nevertheless, the exact mechanism of how the plant immune system shapes

896

microbiome formation remains to be determined.

897

After a successful establishment of symbiosis with rhizobia and mycorrhiza,

898

specifically expressed transporters translocate nutrients between the partners

899[133]. Plants deliver sugars, organic acids, and lipids, and in return, receive

900

phosphate, nitrogen, and other nutrients provided by the microbes [100,128].

901

Compound exchange between plants and rhizobiomes, remains uncharacterized,

902

and we propose plant transporters that could be involved in the process (Table 1,903

see also main text).

904

Figure legends

905

Key Figure 1: Plant and microbial exometabolite networks

906

Plant roots and border cells (brown) and microbes (blue) synthesize metabolites

907

and transporters (boxes), and export certain metabolites into the rhizosphere. This

908

network is depicted with dotted arrows. Exometabolites can have nutritional value909

and signaling functions (solid arrows indicate direction, if the metabolite has only a

910

nutrient or signaling function, the role is specified in brackets). Some microbial

911

epiphytes can migrate from the rhizosphere onto the rhizoplane and into the root,912

where they become endophytes. Plant-microbe and microbe-microbe exometabolite

913

interactions are displayed with numbers: 1, substrate competition between

914

microbes, or between microbes and roots. 2, plant growth promotion by microbial

915

compounds. 3, rhizosphere effect, likely influenced by the presence of916

exometabolites. Plant and microbial exudates are displayed as gradients. Organisms

917

and cells are not to scale.

918
919

Figure 2: Metabolite exchange networks in the rhizosphere

920(A) Flavonoids are exuded, likely by an ABCG-type transporter [123], and sensed by921

rhizobia that in turn produce Nod factors. Rhizobacteria enter the root via root hairs

922

or cracks between epidermal cells [134]. (B) Strigolactones are exuded by ABCG-

923

type Petunia hybrida PDR1 localized in the sub-epidermal layer of the root924

maturation zone [32], and sensed by Glomeromycota that in turn produce Myc

925

factors. Chitin plays a role in hyphal attachment to the root. (C) AtALMT1 is located926

in the cortex of the elongation zone, and is involved in malic acid exudation in

927

Pseudomonas- infected Arabidopsis thaliana, which attracts Bacillus subtilis [81]. B.

928

subtilis form biofilms on roots, a process dependent on root pectin and929

arabinogalactan [46]. (D) ATPases exude protons altering rhizospheric pH, enabling

930

proton dependent transport processes. MATE transporters exude citrate [120],931

which can be metabolized by microbes, and AtPDR9 transports phenolic compounds

932[135]. Signaling function and potential crosstalk with microbes are currently

933

unknown. (E) Involvement of transporters in metabolite exudation is generally

934

poorly understood [27,92,93]. Microbes exude compounds that are utilized by other935

microbes [60,136], and sensed by plants. (F) Border cells produce mucilage (red936

gradient), exude proteins, extracellular DNA, as well as metabolites, all of which937

impact the microbial community [34,41,137]. Currently, the mode of transport of938

these compounds is not characterized.

939

Color legend: blue: microbial components, brown: plant components, red940

transporters:

941

extracellular DNA.

942

characterized,orangetransporters:

uncharacterized.

exDNA:

943

Glossary

944

Antiporter: A transporter utilizing a proton gradient to shuttle protons and945

substrates in opposite directions across a membrane.

946

Apoplasm: intercellular space between plasma membranes of plant tissue.

947

Border cells: root cap cells released into the rhizosphere with a distinct

948

transcriptome, releasing mucilage, distinct proteins, and extracellular DNA.

949

Casparian strip: suberin-based connection between endodermal root cells,

950

blocking passive apoplasmic flow of liquids and compounds.

951

Endophyte: microbe living within plant tissue, in the endosphere.

952

Endosphere: all endophytes of a plant. Relevant to this review are microbes living

953

in root tissues.

954

Epiphyte: microbe living on plant tissue.

955

Exometabolites: compounds released by roots or microbes into the rhizosphere,956

often acting either as nutrients or signaling molecules.

957

Isolate: A microbial strain isolated from a natural environment such as the958

rhizosphere, to be used in a laboratory setting.

959

Mucilage: matrix of high-molecular weight compounds released by border cells and960

root tip.

961

OTU: operational taxonomic unit, often encompassing phylogenetically diverse962

microbes

963

Phytoremediation: accumulation of heavy metals or organic pollutants of964

contaminated soils in plant tissue, with the aim to clean soils.

965

Rhizobiome: the rhizosphere microbiome, consisting of microbes associated with

966

plant roots.

967

Rhizoplane: Root surface including tightly adhered microbes.

968

Rhizosphere: 1-3 mm zone around root shaped by roots, and exudates.

969

Rhizosphere effect: plants with a strong rhizosphere effect have a rhizosphere970

microbiome very distinct from bulk soil. Developmental stage and the type of plant971

species both influence the strength of the rhizosphere effect.

972

Symporter: A transporter utilizing a proton gradient to shuttle protons and973

substrates in the same direction across a membrane.

974

Uniporter: a transporter binding one substrate molecule at a time, facilitating

975

diffusion across a membrane.

976

977
978

Key Figure 1

979

980
981

Figure 2

982

983
984
985

Figure I (Box 2)

986

Table

987
988

Table 1. Transporters for metabolite uptake and releasea

Class
Transport
mode
SugarsImportExportSugar
alcohols
ImportExportSugar
phosphate
Amino acids

ImportExportOrganic
acids
ExportNucleotides
ImportMetabolite examples
Transporter
Description and
Refs.
familylocalization
Glucose, fructose, sucrose, arabinose, xylose, mannose, maltose, ribose, galactose,galactinol, glycerol
MFS (SUC)
Sucrose:H+ symporter, PM [138]
MFS (STP,
Hexose:H+ symporter, PM[83,139] [82]PMT)
SweetsMono- & disaccharides[84,85]
Indirect, vacuolar
MFS (ESL)b
Uniporter?, vacuolar[140]
MFS family
Sugar:H+ antiporter,[86]
Sugar uniporter
Inositol, myo-inositol, threitol, xylitol, erythritol, ribitol
MFS (INT)
MFS (PMT)
MFS (INT)

Inositol:H+ symporter, PM
Polyol:H+ symporter, PM

Inositol:H+ symporter,
indirect, vacuolar
MFS family
H+ antiporter, uniporter
Glucose-6-phosphate, glucose-1-phosphate[139,141][142,143][139][139,143]

Glutamic acid, aspartic acid, alanine, threonine, serine, asparagine, glutamine, valine,
glycine, isoleucine, homoserine, histidine, lysine, arginine, leucine, proline,
phenylalanine, 4-aminobutyric acid, methionine, ornithine, tryptophan, tyrosine
APC (LHT, AAP, Neutral, acidic, basic Aa,
ProT, ANT)PM
[144,145][146]GDU
Glutamine, vasculature, PM [94]Bidirectional Aa in yeast,
[147]
BAT1
PM
Bidirectional Aa in yeast,
[148]
SIAR2
PM
Gamma-aminobutyric acid, [149]CATbidirectional? Vacuolar
UmamiT
Phloem, Aa export, PM[93]
Succinic, malic, tartaric, lactic, formic, butyric, acetic, propionic, gluconic, oxalic, citric,
pyruvic, formic, malonic, a-ketoglutaric, fumaric, trans-aconitic, aspartic, benzoic,
glyceric acid.
ALMT
Malate. Some Al, pathogen [81,150][116]activated, PM
MATE
Citrate. Al, Fe activated.
[151] [152]PM
Adenosine, guanosine, cytidine, thymine
Heterocyclic
Allantoin:H+ symporter,[96]
nitrogen
PM

PUP
ENT
ExportP-type ATPase
ANTMDR (ABC)

PeptidesImport

989
990
991
992
993
994
995

Purine:H+ symporter, PM
Nucleoside, nucleotide.
Some H+ symporter, PM
Extracellular ATP
degradation, indirect, PM
Nucleotide transport, E.
coli, PM
Nucleotide transport, PM

[95]
[153]
[98]
[154]
[97,98]

Oligopeptide:H+ symporter, [155,156]
glutathione, phytochelatins.
PM
PTR
Di-, tripeptide transporter.
[133,156,157]
PM
ExportMDR (ABC)
Peptides, PM[35]
Fatty acids
Linoleic, oleic, palmitic, stearic
Import
P4-ATPase
ATP-dependent flippase,
[158]
(ALA)
PM
ExportABC (PDR,
Lipids for cutin, sterols,[99,100,159-161]
WBC)
mycorrhizal fungi, PM
Inorganics
Nitrate, phosphate, sulfate, potassium
Import
NRT1, NRT2
NO3-/H+ symporter,[156]
high/low affinity. PM
AMT
NH4+. PM?
[133]
MFS (PHT)
Pi/H+ symporter, PM[133,162]
SULTR
Sulfate, PM
[133]
KUP
Potassium, PM[133]
ExportATPaseH+, ATP dependent. PM[163]
Secondary
Coumarins: esculetin, esculin, scopoletin, scopolin, 4-methylumbelliferone.
metabolites, Sterols: campesterol, cholesterol, sitosterol, stigmasterol
hormones
Flavonoids, hormones, glucosinolates
ImportABC (PDR)
Hormones, PM[164,165]
AUX/LAX
Auxin, PM[166]
Hormones, glucosinolates, [164,167]
NRT
PM
ExportABC (PDR,
Hormones, heavy metals.
[35,102][104][32]
MRP, MDR)
ATP dependent, PM
Flavonoids, anthocyanins,[135][168,169]
MATE
xenobiotics, phenolics, PM
Abbreviations: PM, Plasma membrane.
OPT

aAn overview of metabolite classes with examples frequently detected in root exudates, withtransporter families involved in metabolite import (blue) or export (orange). The transporter
function is given in the description section, with localization of the family in roots when not at theplasma membrane.
bText in italic: Additional families likely involved in export of metabolites without experimental
validation