Skip to main content
SpringerLink
Log in

Phyllosphere Metaproteomes of Trees from the Brazilian Atlantic Forest Show High Levels of Functional Redundancy

  • Plant Microbe Interactions
  • Published:
Microbial Ecology Aims and scope Submit manuscript

Abstract

The phyllosphere of the Brazilian Atlantic Forest has been estimated to contain several million bacterial species that are associated with approximately 20000 plant species. Despite the high bacterial diversity in the phyllosphere, the function of these microorganisms and the mechanisms driving their community assembly are largely unknown. In this study, we characterized the bacterial communities in the phyllospheres of four tree species of the Atlantic Forest (Mollinedia schottiana, Ocotea dispersa, Ocotea teleiandra, and Tabebuia serratifolia) and their metaproteomes to examine the basic protein functional groups expressed in the phyllosphere. Bacterial community analyses using 16S rRNA gene sequencing confirmed prior observations that plant species harbor distinct bacterial communities and that plants of the same taxon have more similar communities than more distantly related taxa. Using LC-ESI-Q-TOF, we identified 216 nonredundant proteins, based on 3503 peptide mass spectra. Most protein families were shared among the phyllosphere communities, suggesting functional redundancy despite differences in the species compositions of the bacterial communities. Proteins involved in glycolysis and anaerobic carbohydrate metabolism, solute transport, protein metabolism, cell motility, stress and antioxidant responses, nitrogen metabolism, and iron homeostasis were among the most frequently detected. In contrast to prior studies on crop plants and Arabidopsis, a low abundance of OTUs related to Methylobacterium and no proteins associated with the metabolism of one-carbon molecules were detected in the phyllospheres of the tree species studied here. Our data suggest that even though the phyllosphere bacterial communities of different tree species are phylogenetically diverse, their metaproteomes are functionally convergent with respect to traits required for survival on leaf surfaces.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Lindow SE, Brandl MT (2003) Microbiology of the phyllosphere. Appl Environ Microbiol 69:1875–1883

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Morris C, Kinkel L (2002) Fifty years of phyllosphere microbiology: significant contributions to research in related fields. In: Lindow S, Hecht-Poinar E, Elliott V (eds) Phyllosphere microbiology. APS Press, St. Paul, pp 365–375

    Google Scholar 

  3. Lambais MR, Crowley DE, Cury JC, Büll RC, Rodrigues RR (2006) Bacterial diversity in tree canopies of the Atlantic forest. Science 312:5782

    Article  Google Scholar 

  4. Lambais MR, Lucheta AR, Crowley DE (2014) Bacterial community assemblages associated with the phyllosphere, dermosphere, and rhizosphere of tree species of the Atlantic forest are host taxon dependent. Microb Ecol 68:567–574

    Article  PubMed  Google Scholar 

  5. Vorholt JA (2012) Microbial life in the phyllosphere. Nat Rev Microbiol 10:828–840

    Article  CAS  PubMed  Google Scholar 

  6. Delmotte N, Knief C, Chaffron S, Innerebner G, Roschitzki B, Schlapbach R, Vorholt JA (2009) Community proteogenomics reveals insights into the physiology of phyllosphere bacteria. Proc Natl Acad Sci U S A 106:16428–16433

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Bulgarelli D, Schlaeppi K, Spaepen S, Ver Loren van Themaat E, Schulze-Lefert P (2013) Structure and functions of the bacterial microbiota of plants. Annu Rev Plant Biol 64:807–838

    Article  CAS  PubMed  Google Scholar 

  8. Baker GC, Smith JJ, Cowan DA (2003) Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 55:541–555

    Article  CAS  PubMed  Google Scholar 

  9. Schloss PD, Westcot SL, Ryabin T, Hall JR, Hartman M, Hollister EB (2009) Introducing Mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO (2012) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acid Res 41:D590–D596

    Article  PubMed  PubMed Central  Google Scholar 

  11. Chiu C-H, Chao A (2016) Estimating and comparing microbial diversity in the presence of sequencing errors. Peer J 4:e1634

    Article  PubMed  PubMed Central  Google Scholar 

  12. Wang Q, Garrity GM, Tiedje JM, Cole RJ (2007) Naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lozupone C, Knight R (2005) UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol 71:8228–8235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Keller A, Nesvizhskii AI, Kolker E, Aebersold R (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74:5383–92

    Article  CAS  PubMed  Google Scholar 

  15. Nesvizhskii AI, Keller A, Kolker E, Aebersold R (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75:4646–4658

    Article  CAS  PubMed  Google Scholar 

  16. Vizcaíno JA, Côté RG, Csordas A, Dianes JA, Fabregat A, Foster JM, Griss J, Alpi E, Birim M, Contell J, O’Kelly G, Schoenegger A, Ovelleiro D, Pérez-Riverol Y, Reisinger F, Ríos D, Wang R, Hermjakob H (2013) The Proteomics Identifications (PRIDE) database and associated tools: status in 2013. Nucleic Acid Res 41(Database issue):D1063–D1069

    Article  PubMed  Google Scholar 

  17. Knief C, Delmotte N, Chaffron S, Stark M, Innerebner G, Wassmann R, von Mering C, Vorholt JA (2012) Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice. ISME J 6:1378–1390

    Article  CAS  PubMed  Google Scholar 

  18. Fan L, Reynolds D, Liu M, Stark M, Kjelleberg S, Webster NS, Thomas T (2012) Functional equivalence and evolutionary convergence in complex communities of microbial sponge symbionts. Proc Natl Acad Sci U S A 109:E1878–E1887

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jackson CR, Denney WC (2011) Annual and seasonal variation in the phyllosphere bacterial community associated with leaves of the southern magnolia (Magnolia grandiflora). Microb Ecol 61:113–122

    Article  PubMed  Google Scholar 

  20. Oikawa PY, Giebel BM, Sternberg L, Li L, Timko MP, Swart PK, Riemer DD, Mak JE, Lerdau MT (2011) Leaf and root pectin methylesterase activity and 13C/12C stable isotopic ratio measurements of methanol emissions give insight into methanol production in Lycopersicon esculentum. New Phytol 191:1031–1040

    Article  CAS  PubMed  Google Scholar 

  21. Ofek-Lalzar M, Sela N, Goldman-Voronov M, Green SJ, Hadar Y, Minz D (2014) Niche and host-associated functional signatures of the root surface microbiome. Nat Commun 5:4950

    Article  CAS  PubMed  Google Scholar 

  22. Yuste JC, Fernandez-Gonzales AJ, Fernandez-Lopez M, Ogaya R, Penuelas J, Lloret F (2014) Functional diversification within bacterial lineages promotes wide functional overlapping between taxonomic groups in a Mediterranean forest soil. FEMS Microbiol Ecol 90:54–67

    Article  Google Scholar 

  23. Burke C, Steinberg P, Rusch D, Kjelleberg S, Thomas T (2011) Bacterial community assembly based on functional genes rather than species. Proc Natl Acad Sci U S A 108:14288–14293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhang C, Wei J, Zheng Z, Ying N, Sheng D, Hua Y (2005) Proteomic analysis of Deinococcus radiodurans recovering from γ-irradiation. Proteomics 5:138–143

    Article  CAS  PubMed  Google Scholar 

  25. Leveau JHJ, Lindow SE (2001) Appetite of an epiphyte: quantitative monitoring of bacterial sugar consumption in the phyllosphere. Proc Natl Acad Sci U S A 98:3446–3453

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Weghoff MC, Bertsch J, Müller V (2015) A novel mode of lactate metabolism in strictly anaerobic bacteria. Environ Microbiol 17:670–677

    Article  CAS  PubMed  Google Scholar 

  27. Hinsa SM, Espinosa-Urgel M, Ramos JL, O’Toole GA (2003) Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein. Mol Microbiol 49:905–918

    Article  CAS  PubMed  Google Scholar 

  28. Dunger G, Relling VM, Tondo ML, Barreras M, Ielpi L, Orellano EG, Ottado J (2007) Xanthan is not essential for pathogenicity in citrus canker but contributes to Xanthomonas epiphytic survival. Arch Microbiol 188:127–135

    Article  CAS  PubMed  Google Scholar 

  29. Bogino PC, de L Oliva M, Sorroche FG, Giordano W (2013) The role of bacterial biofilms and surface components in plant-bacterial associations. Int J Mol Sci 14:15838–15859

    Article  PubMed  PubMed Central  Google Scholar 

  30. Milling A, Babujee L, Allen C (2011) Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants. PLoS One 6:e15853

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hopfe M, Dahlmanns T, Henrich B (2011) In Mycoplasma hominis the OppA-mediated cytoadhesion depends on its ATPase activity. BMC Microbiol 11:185

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Krishnan S, Prasadarao N (2012) Outer membrane protein A and OprF—versatile roles in Gram-negative bacterial infection. FBES J 29:997–1003

    Google Scholar 

  33. Gourion B, Rossignol M, Vorholt JA (2006) A proteomic study of Methylobacterium extorquens reveals a response regulator essential for epiphytic growth. Proc Natl Acad Sci U S A 103:13186–13191

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gourion B, Sulser S, Frunzke J, Francez-Charlot A, Stiefel P, Pessi G, Vorholt JA, Fischer HM (2009) The PhyR-sigma (EcfG) signalling cascade is involved in stress response and symbiotic efficiency in Bradyrhizobium japonicum. Mol Microbiol 73:291–305

    Article  CAS  PubMed  Google Scholar 

  35. Santoro MG (2000) Heat shock factors and the control of the stress response. Biochem Pharmacol 59:55–63

    Article  CAS  PubMed  Google Scholar 

  36. Chaikam VL, Karlson DT (2010) Comparison of structure, function and regulation of plant cold shock domain proteins to bacterial and animal cold shock domain proteins. BMB Rep 43:1–8

    Article  CAS  PubMed  Google Scholar 

  37. Fattori J, Prando A, Martins A, Rodrigues FH, Tasic L (2011) Bacterial secretion chaperones. Protein Pept Lett 18:158–166

    Article  CAS  PubMed  Google Scholar 

  38. Fürnkranz M, Wanek W, Richter A, Abell G, Rasche F, Sessitsch A (2008) Nitrogen fixation by phyllosphere bacteria associated with higher plants and their colonizing epiphytes of a tropical lowland rainforest of Costa Rica. ISME J 2:561–570

    Article  PubMed  Google Scholar 

  39. Dangl JL, Horvath DM, Staskawicz BJ (2013) Pivoting the plant immune system from dissection to deployment. Science 341:746–751

    Article  CAS  PubMed  Google Scholar 

  40. Andrews SC, Robinson AK, Rodríguez-Quiñones F (2003) Bacterial iron homeostasis. FEMS Microbiol Rev 27:215–237

    Article  CAS  PubMed  Google Scholar 

  41. Theil EC, Matzapetakis M, Liu X (2006) Ferritins: iron/oxygen biominerals in protein nanocages. J Biol Inorg Chem 11:803–810

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Adriana Franco Paes Leme for making the Mass Spectrometry Facilities at the Brazilian Biosciences National Laboratory (LNBio) available. This project was supported by BIOTA-FAPESP (São Paulo Research Foundation, São Paulo, Brazil) through a grant to MRL.

Author information

Authors and Affiliations

  1. Departamento de Ciência do Solo, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, SP, 13418-900, Brazil

    M. R. Lambais, S. E. Barrera & E. C. Santos

  2. Department of Environmental Sciences, University of California, 900 University Ave, Riverside, CA, 92521, USA

    D. E. Crowley

  3. Division of Biology, Kansas State University, 116 Ackert Hall, Manhattan, KS, 66506, USA

    A. Jumpponen

Authors
  1. M. R. Lambais
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. E. Barrera
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E. C. Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D. E. Crowley
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A. Jumpponen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. R. Lambais.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Table S1

MID sequences used for tagging 16S rRNA gene amplicons. (PDF 21 kb)

Table S2

Relative abundance (%) of OTUs, at the genus level, identified in the phyllospheres of M. schottiana, O. dispersa, O. teleiandra and T. serratifolia. (PDF 246 kb)

Table S3

List of all proteins identified in the phyllosphere of M. schottiana, O. dispersa, O. teleiandra and T. serratifolia. (PDF 261 kb)

Table S4

List of non-redundant proteins identified in the phyllosphere of M. schottiana, O. dispersa, O. teleiandra and T. serratifolia. (PDF 146 kb)

Fig. S1

Geographical locations of individual trees of M. schottiana, O. dispersa, O. teleiandra and T. serratifolia sampled in the Carlos Botelho State Park. For 16S rRNA gene sequencing, sampled trees were located as follows: M. schottiana at sub-plots D07, E00, I09 and H10; O. dispersa at sub-plots D00 and E05; O. teleiandra at sub-plots K05, D02, F07 and M06; T. serratifolia at sub-plots A15, E01, F07, H10 and M09. For protein analyses, sampled trees were located as follows: M. schottiana pool 1 at sub-plots C01, D07, E00 and G02, M. schottiana pool 2 at sub-plots D10, E11, H07 and H10; M. schottiana pool 3 at sub-plots I09, J03, J10 and M11; O. dispersa pool 1 at sub-plots B05, D00, E00 and I01; O. dispersa pool 2 at sub-plots C08, D08, D09 and E05; O. dispersa pool 3 at sub-plots B12, D10, E11 and K12; O. teleiandra pool 1 at sub-plots D02, F05, F07 and I08; O. teleiandra pool 2 at sub-plots J04, J05, K05 and L11; O. teleiandra pool 3 at sub-plots K13, L00, L01 and M06; T. serratifolia pool 1 at sub-plots A05, E01, E02 and J04; T. serratifolia pool 2 at sub-plots E06, F07, H10 and H11; T. serratifolia pool 3 at sub-plots A15, E12, J08 and M09. Numbers on the outer square represent the UTM coordinates. Numbers and letters on the inner square represent the sub-plot coordinates. (GIF 27 kb)

High resolution image (TIF 3141 kb)

Fig. S2

Rarefaction curves for each tree species analyzed. Operational taxonomic units (OTUs) were assigned at 97 % similarity. Bars represent the 95 % confidence intervals. (GIF 31 kb)

High resolution image (TIF 29991 kb)

Fig. S3

Relative abundance of bacterial classes identified in the phyllospheres of M. schottiana, O. dispersa, O. teleiandra and T. serratifolia trees in the Atlantic Forest. Data represent the mean per species group. Bars represent the 95 % confidence intervals. (GIF 191 kb)

High resolution image (TIF 88495 kb)

Fig. S4

Profiles of Pfam domains identified in the metaproteomes of the phyllospheres of M. schottiana, O. dispersa, O. teleiandra and T. serratifolia trees in the Atlantic Forest, soybean, clover, Arabidopsis and rice. Except for rice, all the profiles were based on the 20 most abundant proteins in the phyllosphere. In rice, profile was based on the 20 most abundant Pfam domains enriched in the phyllosphere. Data represent Pfams detected (red) or not detected (blank). (GIF 102 kb)

High resolution image (TIF 2693 kb)

Fig. S5

Venn’s diagram based on the Pfam domains identified in the phyllospheres of M. schottiana, O. dispersa, O. teleiandra and T. serratifolia trees in the Atlantic Forest. (GIF 380 kb)

High resolution image (TIF 116 kb)

Fig. S6

Principal coordinates plots based on the abundance of Pfam domains identified in the phyllospheres of M. schottiana, O. dispersa, O. teleiandra and T. serratifolia trees. The values in parenthesis associated with the PCo1 and PCo2 represent the proportion of the variance explained by each component. Ellipse represents 95 % confidence. (GIF 144 kb)

High resolution image (TIF 2932 kb)

Fig. S7

Protein based taxonomy of the bacterial groups in the phyllospheres of M. schottiana, O. dispersa, O. teleiandra and T. serratifolia. (GIF 52 kb)

High resolution image (TIF 1100 kb)

Fig. S8

Unmatched peptide based taxonomy of the bacterial groups in the phyllospheres of M. schottiana, O. dispersa, O. teleiandra and T. serratifolia. (GIF 3325 kb)

High resolution image (TIF 1991 kb)

Fig. S9

Relative abundances of the different bacterial taxa at the class level based on 16S rRNA gene and protein sequences. Data for M. schottiana, O. dispersa, O. teleiandra and T. serratifolia are based in this study, and for rice are based on Knief et al. [17]. (GIF 30 kb)

High resolution image (TIF 35861 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lambais, M.R., Barrera, S.E., Santos, E.C. et al. Phyllosphere Metaproteomes of Trees from the Brazilian Atlantic Forest Show High Levels of Functional Redundancy. Microb Ecol 73, 123–134 (2017). https://doi.org/10.1007/s00248-016-0878-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00248-016-0878-6

Keywords

Search

Navigation