Root exudates drive interspecific facilitation by enhancing nodulation and N<sub>2</sub> fixation

Bai Li

Yu Li

Hua Wu

Fang Zhang

^{Key Laboratory of Plant–Soil Interactions, Ministry of Education, Beijing Key Laboratory of Biodiversity and Organic Farming, College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, People’s Republic of China;}

^{School of Plant Biology and Institute of Agriculture, University of Western Australia, Crawley, Perth, WA 6009, Australia}

^{To whom correspondence should be addressed. Email:}nc.ude.uac@gnolil.

Edited by David Tilman, University of Minnesota, St. Paul, MN, and approved April 29, 2016 (received for review November 29, 2015)

Author contributions: B.L., X.-X.L., and L.L. designed research; B.L., Y.-Y.L., H.-M.W., F.-F.Z., C.-J.L., and L.L. performed research; B.L., Y.-Y.L., X.-X.L., and L.L. analyzed data; and B.L., X.-X.L., H.L., and L.L. wrote the paper.

^{Present address: Clinical Laboratory, Sentara Martha Jefferson Hospital, Charlottesville, VA 22911.}

^{Present address: College of Agriculture and Engineering, Nanyang Normal University, Nanyang 473061, Henan Province, China.}

^{Present address: Crop Production Service Center of Yanqing County, Beijing 102100, China.}

Edited by David Tilman, University of Minnesota, St. Paul, MN, and approved April 29, 2016 (received for review November 29, 2015)

Freely available online through the PNAS open access option.

Significance

Plant diversity often leads to an increase in ecosystem productivity, but the underpinning mechanisms remain poorly understood. We found that faba bean/maize intercropping enhances productivity, nodulation, and N₂ fixation of faba bean through interspecific root interactions. We provide a mechanism explaining how maize promotes N₂ fixation of faba bean, where root exudates from maize increase root hair deformation and nodulation in faba bean, double exudation of flavonoids (signaling compounds for rhizobia), and up-regulate the expression of a chalcone–flavanone isomerase gene involved in flavonoid synthesis, and genes mediating nodulation and auxin responses. Our results provide a mechanism for facilitative root–root interactions explaining how species diversity may enhance ecosystem productivity with important implications for developing sustainable agriculture.

Keywords: flavanoids, gene expression, intercropping, root–root interactions, signals

Significance

Abstract

Plant diversity in experimental systems often enhances ecosystem productivity, but the mechanisms causing this overyielding are only partly understood. Intercropping faba beans (Vicia faba L.) and maize (Zea mays L.) result in overyielding and also, enhanced nodulation by faba beans. By using permeable and impermeable root barriers in a 2-y field experiment, we show that root–root interactions between faba bean and maize significantly increase both nodulation and symbiotic N₂ fixation in intercropped faba bean. Furthermore, root exudates from maize promote faba bean nodulation, whereas root exudates from wheat and barley do not. Thus, a decline of soil nitrate concentrations caused by intercropped cereals is not the sole mechanism for maize promoting faba bean nodulation. Intercropped maize also caused a twofold increase in exudation of flavonoids (signaling compounds for rhizobia) in the systems. Roots of faba bean treated with maize root exudates exhibited an immediate 11-fold increase in the expression of chalcone–flavanone isomerase (involved in flavonoid synthesis) gene together with a significantly increased expression of genes mediating nodulation and auxin response. After 35 d, faba beans treated with maize root exudate continued to show up-regulation of key nodulation genes, such as early nodulin 93 (ENOD93), and promoted nitrogen fixation. Our results reveal a mechanism for how intercropped maize promotes nitrogen fixation of faba bean, where maize root exudates promote flavonoid synthesis in faba bean, increase nodulation, and stimulate nitrogen fixation after enhanced gene expression. These results indicate facilitative root–root interactions and provide a mechanism for a positive relationship between species diversity and ecosystem productivity.

Abstract

Many ecosystems, including grasslands (1, 2), forests (3), phytoplankton communities (4), and cropping systems (5), show a positive relationship between plant diversity and ecosystem productivity. Several mechanisms have been proposed to explain this relationship. A “sampling effect” occurs, because more diverse mixtures have a higher probability of containing a species with higher productivity (6). Complementarity effects occur when species vary in resource use and niche differentiation in space or time, leading to greater resource utilization (6 –9). Facilitation occurs when one species increases the growth of other species through a wide range of processes (10). Facilitative effects may be direct (e.g., by shade or protection from harsh conditions) or indirect (e.g., when one species reduces attack by pathogens or herbivores on other species) (11 –13).

Legume/cereal intercropping systems have been widely studied in the context of diversity and ecosystem function and commonly overyield, because dinitrogen (N₂) fixation by legumes increases ecosystem nitrogen (N) supply (7, 8), an example of facilitation. This facilitation is important to agriculture on a large scale, because application of chemical N fertilizer decreases biological N₂ fixation by legumes. Intercropping with maize increases N₂ fixation by faba bean, even with high input of N fertilizer (7), but such a stimulatory effect on faba bean has not been observed in all legume/cereal intercropping systems (7), suggesting species-specific facilitative relationships (14). Plants communicate by an “underground highway”—root exudates, which inhibit or facilitate their neighbors (15)—and part of this communication is through flavonoids, which are key signals in nodulation of legumes. How exactly maize promotes faba bean nodulation and the potential role of flavonoids remain unclear. Here, we explore cross-talk between maize and faba bean through rhizosphere processes and physiological and molecular mechanisms underpinning this communication.

Values for grain yield are averages of 2 y (2006 and 2007). Values in the same row followed by different letters are significantly different (P < 0.05).

Acknowledgments

The authors thank Dr. Ragan Callaway and Jacob Lucero for their valuable comments and editing the manuscript and Prof. Wenxin Chen for providing rhizobium (strain NM353) inoculum. This work was supported by National Science Foundation of China Grants 30670381 and 31430014 and National Basic Research Programme of China 973 Program Grant 2011CB100405.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the NCBI database (accession nos. {"type":"entrez-nucleotide","attrs":{"text":"KU973538","term_id":"1033939007","term_text":"KU973538"}}KU973538–{"type":"entrez-nucleotide","attrs":{"text":"KU973547","term_id":"1033939025","term_text":"KU973547"}}KU973547).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1523580113/-/DCSupplemental.

Footnotes

References

1. Hector A, et al Plant diversity and productivity experiments in european grasslands. Science. 1999;286(5442):1123–1127.[PubMed][Google Scholar]
2. Tilman D, et al Diversity and productivity in a long-term grassland experiment. Science. 2001;294(5543):843–845.[PubMed][Google Scholar]
3. Lovelock CE, Ewel JJLinks between tree species, symbiotic fungal diversity and ecosystem functioning in simplified tropical ecosystems. New Phytol. 2005;167(1):219–228.[PubMed][Google Scholar]
4. Ptacnik R, et al Diversity predicts stability and resource use efficiency in natural phytoplankton communities. Proc Natl Acad Sci USA. 2008;105(13):5134–5138.[Google Scholar]
5. Huston MAHidden treatments in ecological experiments: Re-evaluating the ecosystem function of biodiversity. Oecologia. 1997;110(4):449–460.[PubMed][Google Scholar]
6. Hector A, Bazeley-White E, Loreau M, Otway S, Schmid BOveryielding in grassland communities: Testing the sampling effect hypothesis with replicated biodiversity experiments. Ecol Lett. 2002;5(4):502–511.[PubMed][Google Scholar]
7. Fan FL, et al Nitrogen fixation of faba bean (Vicia faba L.) interacting with a non-legume in two contrasting intercropping systems. Plant Soil. 2006;283(1):275–286.[PubMed][Google Scholar]
8. Li YY, et al Intercropping alleviates the inhibitory effect of N fertilization on nodulation and symbiotic N₂ fixation of faba bean. Plant Soil. 2009;323(1):295–308.[PubMed][Google Scholar]
9. Fargione J, Tilman DNiche differences in phenology and rooting depth promote coexistence with a dominant C₄ bunchgrass. Oecologia. 2005;143(4):598–606.[PubMed][Google Scholar]
10. Callaway RM Positive Interactions and Interdependence in Plant Communities. Springer; Berlin: 2007. [PubMed][Google Scholar]
11. Knops JMH, et al Effects of plant species richness on invasion dynamics, disease outbreaks, insect abundances and diversity. Ecol Lett. 1999;2(5):286–293.[PubMed][Google Scholar]
12. Maron JL, Marler M, Klironomos JN, Cleveland CCSoil fungal pathogens and the relationship between plant diversity and productivity. Ecol Lett. 2011;14(1):36–41.[PubMed][Google Scholar]
13. Schnitzer SA, et al Soil microbes drive the classic plant diversity-productivity pattern. Ecology. 2011;92(2):296–303.[PubMed][Google Scholar]
14. Callaway RMAre positive interactions species-specific? Oikos. 1998;82(1):202–207.[PubMed][Google Scholar]
15. Callaway RMThe detection of neighbors by plants. Trends Ecol Evol. 2002;17(3):104–105.[PubMed][Google Scholar]
16. Spaink HPRoot nodulation and infection factors produced by rhizobial bacteria. Annu Rev Microbiol. 2000;54:257–288.[PubMed][Google Scholar]
17. Jensen ESGrain yield, symbiotic N₂ fixation and interspecific competition for inorganic N in pea-barley intercrops. Plant Soil. 1996;182(1):25–38.[PubMed][Google Scholar]
18. Li QZ, et al Overyielding and interspecific interactions mediated by nitrogen fertilization in strip intercropping of maize with faba bean, wheat and barley. Plant Soil. 2011;339(1):147–161.[PubMed][Google Scholar]
19. Miao R, Zhang FS, Li LEffects of root barriers on nodulation of faba bean in maize/faba bean, wheat/faba bean and barley/faba bean intercropping systems. Chinese Bulletin of Botany. 2009;44(2):197–201.[PubMed][Google Scholar]
20. Zaat SAJ, van Brussel AAN, Tak T, Pees E, Lugtenberg BJJFlavonoids induce Rhizobium leguminosarum to produce nodDABC gene-related factors that cause thick, short roots and root hair responses on common vetch. J Bacteriol. 1987;169(7):3388–3391.[Google Scholar]
21. Müller J, Staehelin C, Xie ZP, Neuhaus-Url G, Boller TNod factors and chitooligomers elicit an increase in cytosolic calcium in aequorin-expressing soybean cells. Plant Physiol. 2000;124(2):733–740.[Google Scholar]
22. Esseling JJ, Lhuissier FGP, Emons AMCA nonsymbiotic root hair tip growth phenotype in NORK-mutated legumes: Implications for nodulation factor-induced signaling and formation of a multifaceted root hair pocket for bacteria. Plant Cell. 2004;16(4):933–944.[Google Scholar]
23. Barcelo J, Poschenrieder CFast root growth responses, root exudates, and internal detoxification as clues to the mechanisms of aluminium toxicity and resistance: A review. Environ Exp Bot. 2002;48(1):75–92.[PubMed][Google Scholar]
24. Lian B, Souleimanov A, Zhou X, Smith DLIn vitro induction of lipo-chitooligosaccharide production in Bradyrhizobium japonicum cultures by root extracts from non-leguminous plants. Microbiol Res. 2002;157(3):157–160.[PubMed][Google Scholar]
25. Zaat SAJ, Schripsema J, Wijffelman CA, van Brussel AAN, Lugtenberg BJJAnalysis of the major inducers of the Rhizobium nodA promoter from Vicia sativa root exudate and their activity with different nodD genes. Plant Mol Biol. 1989;13(2):175–188.[PubMed][Google Scholar]
26. Maj D, Wielbo J, Marek-Kozaczuk M, Skorupska AResponse to flavonoids as a factor influencing competitiveness and symbiotic activity of Rhizobium leguminosarum. Microbiol Res. 2010;165(1):50–60.[PubMed][Google Scholar]
27. Robbins MP, Dixon RA. Induction of chalcone isomerase in elicitor-treated bean cells. Comparison of rates of synthesis and appearance of immunodetectable enzyme. Eur J Biochem. 1984;145(1):195–202.[PubMed]
28. Liu Z, et al Regulation, evolution, and functionality of flavonoids in cereal crops. Biotechnol Lett. 2013;35(11):1765–1780.[PubMed][Google Scholar]
29. Scheres B, et al Sequential induction of nodulin gene expression in the developing pea nodule. Plant Cell. 1990;2(8):687–700.[Google Scholar]
30. Lambers H, Chapin FS, Pons TL Plant Physiological Ecology. 2nd Ed Springer; New York: 2008. [PubMed][Google Scholar]
31. Walker TS, Bais HP, Grotewold E, Vivanco JMRoot exudation and rhizosphere biology. Plant Physiol. 2003;132(1):44–51.[Google Scholar]
32. Shaw LJ, Morris P, Hooker JEPerception and modification of plant flavonoid signals by rhizosphere microorganisms. Environ Microbiol. 2006;8(11):1867–1880.[PubMed][Google Scholar]
33. Abdel-Lateif K, Bogusz D, Hocher VThe role of flavonoids in the establishment of plant roots endosymbioses with arbuscular mycorrhiza fungi, rhizobia and Frankia bacteria. Plant Signal Behav. 2012;7(6):636–641.[Google Scholar]
34. Hassan S, Mathesius UThe role of flavonoids in root-rhizosphere signalling: Opportunities and challenges for improving plant-microbe interactions. J Exp Bot. 2012;63(9):3429–3444.[PubMed][Google Scholar]
35. Weston LA, Mathesius UFlavonoids: Their structure, biosynthesis and role in the rhizosphere, including allelopathy. J Chem Ecol. 2013;39(2):283–297.[PubMed][Google Scholar]
36. Brundrett MCMycorrhizal associations and other means of nutrition of vascular plants: Understanding the global diversity of host plants by resolving conflicting information and developing reliable means of diagnosis. Plant Soil. 2009;320(1):37–77.[PubMed][Google Scholar]
37. Li L, et al Root distribution and interactions between intercropped species. Oecologia. 2006;147(2):280–290.[PubMed][Google Scholar]
38. Shearer G, Kohl DHN₂-fixation in field settings: Estimations based on natural ^{N abundance.}Aust J Plant Physiol. 1986;13(6):699–756.[PubMed][Google Scholar]
39. Boddey RM, Peoples MB, Palmer B, Dart PJUse of the N-15 natural abundance technique to quantify biological nitrogen fixation by woody perennials. Nutrient Cycling in Agroecosystems. 2000;57(3):235–270.[PubMed][Google Scholar]
40. Fåhraeus GThe infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol. 1957;16(2):374–381.[PubMed][Google Scholar]
41. Heidstra R, et al Root hair deformation activity of nodulation factors and their fate on vicia-sativa. Plant Physiol. 1994;105(3):787–797.[Google Scholar]
42. Yashima H, et al Systemic and local effects of long-term application of nitrate on nodule growth and n-2 fixation in soybean (glycine max) Soil Science and Plant Nutrition. 2003;49(6):825–834.[PubMed][Google Scholar]
43. Schmittgen TD, Livak KJAnalyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3(6):1101–1108.[PubMed][Google Scholar]