Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Environ Microbiol. Author manuscript; available in PMC 2013 March 26.
Published in final edited form as:
PMCID: PMC3608849

Microbial rhodopsins on leaf surfaces of terrestrial plants


The above-ground surfaces of terrestrial plants, the phyllosphere, comprise the main interface between the terrestrial biosphere and solar radiation. It is estimated to host up to 1026 microbial cells that may intercept part of the photon flux impinging on the leaves. Based on 454-pyrosequencing-generated metagenome data, we report on the existence of diverse microbial rhodopsins in five distinct phyllospheres from tamarisk (Tamarix nilotica), soybean (Glycine max), Arabidopsis (Arabidopsis thaliana), clover (Trifolium repens) and rice (Oryza sativa). Our findings, for the first time describing microbial rhodopsins from non-aquatic habitats, point towards the potential coexistence of microbial rhodopsin-based phototrophy and plant chlorophyll-based photosynthesis, with the different pigments absorbing non-overlapping fractions of the light spectrum.


Solar radiation is the main source of energy for both marine and terrestrial organisms, with terrestrial plants and aquatic phytoplankton performing an equivalent ecological function as chlorophyll-based photosynthetic primary producers (Field et al., 1998). Marine surface waters are now known to harbour an additional type of phototrophy; several lineages of bacteria and archaea utilize rhodopsins (Béjà et al., 2000; 2001; de la Torre et al., 2003; Balashov et al., 2005; Giovannoni et al., 2005; Sabehi et al., 2005; Frigaard et al., 2006; Gómez-Consarnau et al., 2007; 2010; Oh et al., 2010), retinal-containing transmembrane proteins, as light-driven proton pumps. The first microbial rhodopsin was reported four decades ago in the archaeon Halo-bacterium salinarum from hypersaline environments (Oesterhelt and Stoeckenius, 1971). Further studies revealed the existence of microbial rhodopsins in diverse habitats including freshwater, sea ice, hypersaline and brackish environments (Rusch et al., 2007; Atamna-Ismaeel et al., 2008; Sharma et al., 2008; 2009; Koh et al., 2010). To date, microbial rhodopsins have been reported exclusively for aquatic habitats.

As light is an abundant resource on land, we tested the hypothesis that microbial rhodopsins also exist and play an important role in terrestrial niches. The leaf surface of terrestrial plants covers a surface area of an estimated 6.4 × 108 km2 and comprises the main interface between terrestrial biomass and solar photon flux. This habitat harbours an immensely diverse microbial community of up to 106–107 cells per cm2 leaf surface (Lindow and Brandl, 2003). A mode of phototrophy that is compatible with the plant’s photosynthesis would offer a significant ecological advantage to microbes inhabiting this environment.

Results and discussion

We have identified 156 microbial rhodopsin sequences in five phyllosphere metagenomes (Supporting material S1, S2, S3, S4 and S5 in Supporting information), from different terrestrial plants: soybean (Glycine max) (Delmotte et al., 2009), tamarisk (Tamarix nilotica), clover (Trifolium repens), rice (Oryza sativa), and from a wild population of the model plant Arabidopsis thaliana. The size of the different metagenomes obtained was 261 Mb, 448 Mb, 234 Mb, 831 Mb and 250 Mb for soybean, tamarisk, clover, rice and Arabidopsis with an average read length of 235, 328, 235, 357 and 233 bp, respectively.

Phylogenetic analysis revealed that some phyllosphere microbial rhodopsins have branched away from known rhodopsin families within the bacterial and eukaryal domains (Fig. 1). Some of these sequences clustered with fungal rhodopsins, while another group clustered with xanthorhodopsins (Balashov et al., 2005; Lanyi and Balashov, 2008) and actinorhodopsins (Sharma et al., 2008; 2009). However, most phyllosphere rhodopsins appear on novel branches, with no representatives from either culture-based or environmental data sets, thus rendering them with an as yet uncertain phylogenetic affiliation. In most cases, the leaf surface rhodopsins from tamarisk clustered separately from other phyllosphere rhodopsins (Fig. 1) with a statistically significant phylogenetic signal [calculated using Mesquite (Maddison and Maddison, 2010)], indicating that they reside in distinct microbial taxa, probably adapted to the unique hypersaline environment of the tamarisk phyllosphere (Qvit-Raz et al., 2008).

Fig. 1
A phylogenetic tree of rhodopsin amino acid sequences (deduced from the metagenomic data) from the phyllospheres of tamarisk, rice, soybean, Arabidopsis and clover. Following alignment computation (see Experimental procedures), a FastTree version 2.1.1 ...

In contrast with soil metagenomes, which do not contain any rhodopsin reads, the five phyllosphere data sets were found to contain microbial rhodopsins, but at frequencies lower than those found in marine and fresh-water metagenomes (Fig. 2). While some of the phyllosphere rhodopsins lack the retinylidene Schiff base proton donor carboxylate and are thus likely sensory rhodopsins, others contain both proton acceptor and donor carboxylates at helix C (bacteriorhodopsin positions 85 and 96 respectively; see Supporting material S1, S2, S3, S4 and S5) and may be considered as potential proton pumps. Compared with the marine environment, where they make up only 3% of all microbial rhodopsins (Spudich, 2006), the contribution of sensory rhodopsins to phyllospheres is much higher (25–70%; Fig. 3). This suggests that microorganisms in the phyllosphere are intensively engaged in light sensing, to accommodate the effects of fluctuations in light quality, intensity and UV radiation at the leaf surface (Ballaré et al., 1990; Beattie and Lindow, 1999).

Fig. 2
Relative abundance of microbial rhodopsins in different metagenomes. MG-RAST (Meyer et al., 2008) accession numbers of the different data sets can be found in Experimental procedures. Abundance was normalized relative to the numbers of rplA, rplC, rplD ...
Fig. 3
Sensory rhodopsins and proton pumps in different environments. Proportions of sensory rhodopsins and rhodopsin proton pumps were calculated only from reads containing the region surrounding the proton acceptor and donor carboxylates at helix C (bacteriorhodopsin ...

Interestingly, all detected phyllosphere rhodopsins carry a leucine residue at position 105 (Fig. 4; based on sequence reads that contain this region; not all reads cover the entire gene), which renders them as putative green light absorbing pigments (Man et al., 2003), thus avoiding an overlap with the absorption spectrum of the plant’s leaf and possibly even masking out the negative role of green light on plant growth (Folta and Maruhnich, 2007). This is opposed to blue light absorbing rhodopsins (Béjà et al., 2001; Sabehi et al., 2005), which contain a glutamine instead of leucine at position 105, and are abundant in marine habitats (Béjà et al., 2001; Rusch et al., 2007; Sabehi et al., 2007).

Fig. 4
Protein alignment of phyllosphere rhodopsins. Amino acid position 105 is marked with green or blue backgrounds according to the predicted absorption spectra of the rhodopsin pigments. Only the vicinity of amino acid 105 is shown. Examples from confirmed ...

Another indication that this may indeed be the case in the tamarisk phyllosphere is presented by the absorption spectra in Fig. 5; it is demonstrated that the microbes washed off the leaves have an absorption maximum around 545 nm, a region of the spectrum where there is no light absorption by the tamarisk leaves and where the absorption of microbial rhodopsins is maximal. This absorption peak, however, could also be the result of the presence of carotenoids-containing pink-pigmented Methylobacterium spp. (Kutschera, 2007) in the leaf wash.

Fig. 5
Absorbance spectra of tamarisk leaves and phyllosphere wash. Absorbance of tamarisk chlorophylls (acetone extract) and of phyllosphere leaf buffer-wash are shown; note the different scales used.

This is the first report on the existence of microbial rhodopsins in terrestrial habitats; whether it portrays commensalism or mutualism should be a matter of further investigations. We show that rhodopsin sequences have been found to be abundant both in the harsh environment of the tamarisk phyllosphere (Qvit-Raz et al., 2008) and on the leaves of cultivated plants; furthermore, they are common to diverse leaf shapes and plant growth characteristics, but are absent from both agricultural and forest soils. This indicates that microbial rhodopsins may be selected for in the phyllosphere environment, thus conferring an important adaptive trait onto this microbial niche. We propose that rhodopsin light interception by phyllosphere bacteria needs to be taken into account in global energy balance and biomass production by the terrestrial biosphere.

Experimental procedures

Phyllosphere sampling

Leaf samples were collected from a T. nilotica tree in an oasis by the Dead Sea (31°42′41.06″N 35°27′19.32″E), and processed within 1 h of sampling (Qvit-Raz et al., 2008). Briefly, 50 g of leaves were placed inside a 250 ml sterile glass Erlenmeyer flask, immediately immersed in sterile phosphate-buffered saline (1 g leaf per 5 ml PBS, pH 7.4), and cavitated in a sonication bath [Transistor/ultrasonic T7 (L&R Manufacturing Company)] for 2 min at medium intensity. The preparations were then vortexed 6 × 10 s at 5 min intervals, and the leaf wash was separated from the leaf debris by decanting and kept for analysis. Arabidopsis, clover and rice phyllospheres were prepared according to the previously reported soybean phyllosphere preparation (Delmotte et al., 2009).

DNA extraction and pyrosequencing

The leaf wash was filtered on a 0.22 μm membrane filter (Millipore), which was subjected to total community DNA extraction, using a Power Soil Microbial DNA extraction kit (MoBio). Sequencing was performed on the Genome Sequencer FLX system using 3 μg of DNA at a concentration of 17 ng μl−1 (as determined by a NanoDrop spectrophotometer). The resulting reads were annotated using the MG-RAST rapid annotation platform (Meyer et al., 2008). Using this platform, rhodopsin-containing reads were located within each of the compared metagenomes using an e-value cutoff of 10−5. For the phylogenetic analysis, hits with higher e-values were included as well. The number of reads was normalized against the average number of selected single-copy genes found in the data sets using an e-value cut-off of 10−20.

All non-phyllosphere datasets used are publicly available on the MG-RAST website. The soybean phyllosphere metagenome can be found in the GenBank SRA database. The rhodopsin-containing reads from the phyllosphere metagenomes are provided in Supporting material S1, S2, S3, S4 and S5 in Supporting information.

Phylogenetic tree analysis

In this work, we tried several methods for multiple sequence alignment calculation [MUSCLE, ProbCons, MAFFT and PROMALS, see references within Kemena and Notredame (2009)]. In an effort to automatically identify the most reliable multiple sequence alignment for a given protein family, we used the AQUA protocol for automated quality improvement of multiple sequence alignments (Muller et al., 2010). We performed several alignments using MUSCLE, MAFFT, Prob-Cons, along with one refinement program (RASCAL) and one assessment program (NORMD). According to this protocol the MAFFT alignment refined by RASCAL produced the most reliable alignment (highest NORMD value) and was used to produce the phylogenetic tree. Following the alignment computation, we used FastTree version 2.1.1 SSE3 (Price et al., 2009) for the calculation of the phylogenetic tree using settings for high accuracy [-spr 4 (to increase the number of rounds of minimum-evolution SPR moves) and -mlacc 2 -slownni (to make the search for the most likely alternative topology more exhaustive)]. These parameters can produce slight increases in accuracy. To estimate the reliability of each branching point, FastTree uses a Shimodaire-Hasegawa test on the three alternative topologies (NNIs) around that split (Guindon et al., 2009). Phyllogenetic protein trees were visualized and edited using Dendroscope software version 2.7.3 (Huson et al., 2007).

To test if the phylogenetic signal we observe is statistically significant we used the Mesquite program (Maddison and Maddison, 2010). This was done using a randomization test (to see if the observed number of changes on the tree is less than 95% of the null values). The 10 000 reshufflings of the characters (five different plants and other environments) allowed constructing a character chart of parsimonious changes between the six characters assigned.

Relative abundance of microbial rhodopsins in different metagenomes

Frequency of rhodopsin blast hits with an e-value ≤ 10−5 was determined for 14 metagenomes from phyllosphere (5), marine (5), freshwater (1), hypersaline (1) and soil (2) environments. Rhodopsin abundance was normalized with the abundance of rplA, rplC, rplD, rpoA, rpoB and rspJ genes (Frank and Sorensen, 2011) (blast hits with an e-value ≤ 1e-20) according to Yutin and colleagues (2007) and Howard and colleagues (2008).

Metagenomic data sets used for comparison (Fig. 2)

Freshwater: GS020, Lake Gatun, Panama (MG_RAST accession: 4441590.3)

Hypersaline: GS033, Punta Cormorant hypersaline lagoon, Galapagos (MG-RAST accession: 4441599.3)

Open Sea: GS000a, Sargasso Station 11 (MG-RAST accession: 4441570.3) and GS000b, Sargasso Station 13 (MG-RAST accession: 4441573.3)

Estuary: Monterey Bay (MG-RAST accession: 4443712.3)

Whale Fall: Whale fall Bone (MG-RAST accession: 4441619.3)

Forest Soil: Luquillo experimental forest soil, Puerto Rico (MG-RAST accession: 4446153.3) and Waseca farm soil (MG-RAST accession: 4441091.3)

Soybean: SRA accession: SRX008324 (

Absorbance spectra of tamarisk leaves and phyllosphere wash (Fig. 5)

Phyllosphere absorbance was calculated as the difference between two measurements of reflectance spectra (intact tamarisk leaves and phosphate-buffered saline washed, sonicated leaves), obtained at room temperature with a Labsphere DRA-CA-30I diffuse reflectance accessory. Leaves were densely arranged on a slide and covered with another slide. Two empty slides were used as a blank. Measurements were performed on four different leaf samples from different dates. For chlorophyll absorbance, tamarisk leaves were ground with 90% acetone and filtered through GFF filters. The extract was measured using a Cary 100 spectrophotometer.

Supplementary Material

Suppl Data S1-S5


We thank Robert Edgar for his help and discussions over issues of short sequence alignments and Eli Geffen for his help with statistics. Our gratitude to the staff and scientists of the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution at the Marine Biology Laboratory (Woods Hole, MA, USA) for their help. This work was supported in part by a grant from Bridging the Rift Foundation (O.B. and S.B.), Israel Science Foundation grant 1203/06 (O.B.), the Gruss-Lipper Family Foundation at MBL (O.M.F., S.B. and A.F.P.), a US-Israel Binational Science Foundation grant 2006324 (S.B.), and National Institutes of Health Grant R37GM27750, Department of Energy Grant DE-FG02-07ER15867, and endowed chair AU-0009 from the Robert A. Welch Foundation (J.L.S.).


Supporting information

Additional Supporting Information may be found in the online version of this article:

Supporting material S1. Tamarix phyllosphere microbial rhodopsin sequences.

Supporting material S2. Soybean phyllosphere microbial rhodopsin sequences.

Supporting material S3. Rice phyllosphere microbial rhodopsin sequences.

Supporting material S4. Arabidopsis phyllosphere microbial rhodopsin sequences.

Supporting material S5. Clover phyllosphere microbial rhodopsin sequences.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.


  • Atamna-Ismaeel N, Sabehi G, Sharon I, Witzel K-P, Labrenz M, Jürgens K, et al. Widespread distribution of proteorhodopsins in freshwater and brackish ecosystems. ISME J. 2008;2:656–662. [PubMed]
  • Balashov SP, Imasheva ES, Boichenko VA, Anton J, Wang JM, Lanyi JK. Xanthorhodopsin: a proton pump with a light-harvesting carotenoid antenna. Science. 2005;309:2061–2064. [PMC free article] [PubMed]
  • Ballaré CL, Scopel AL, Sánchez RA. Far-red radiation reflected from adjacent leaves: an early signal of competition in plant canopies. Science. 1990;247:329–332. [PubMed]
  • Beattie GA, Lindow SE. Bacterial colonization of leaves: a spectrum of strategies. Phytopathol. 1999;89:353–359. [PubMed]
  • Béjà O, Aravind L, Koonin EV, Suzuki MT, Hadd A, Nguyen LP, et al. Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science. 2000;289:1902–1906. [PubMed]
  • Béjà O, Spudich EN, Spudich JL, Leclerc M, DeLong EF. Proteorhodopsin phototrophy in the ocean. Nature. 2001;411:786–789. [PubMed]
  • Delmotte N, Knief C, Chaffron S, Innerebner G, Roschitzki B, Schlapbach R, et al. Community proteogenomics reveals insights into the physiology of phyllosphere bacteria. Proc Natl Acad Sci USA. 2009;106:16428–16433. [PubMed]
  • Field CB, Behrenfeld MJ, Randerson JT, Falkowski P. Primary production of the biosphere: integrating terrestrial and oceanic components. Science. 1998;281:237–240. [PubMed]
  • Folta KM, Maruhnich SA. Green light: a signal to slow down or stop. J Exp Bot. 2007;58:3099–3111. [PubMed]
  • Frank JA, Sorensen SJ. Quantitative metagenomic analyses based on average genome size normalization. Appl Environ Microbiol. 2011;77:2513–2521. [PMC free article] [PubMed]
  • Frigaard N-U, Martinez A, Mincer TJ, DeLong EF. Proteorhodopsin lateral gene transfer between marine planktonic Bacteria and Archaea. Nature. 2006;439:847–850. [PubMed]
  • Giovannoni SJ, Bibbs L, Cho J-C, Stapels MD, Desiderio R, Vergin KL, et al. Proteorhodopsin in the ubiquitous marine bacterium SAR11. Nature. 2005;438:82–85. [PubMed]
  • Gómez-Consarnau L, González JM, Coll-Lladó M, Gourdon P, Pascher T, Neutze R, et al. Light stimulates growth of proteorhodopsin-containing marine Flavobacteria. Nature. 2007;445:210–213. [PubMed]
  • Gómez-Consarnau L, Akram N, Lindell K, Pedersen A, Neutze R, Milton DL, et al. Proteorhodopsin phototrophy confers enhanced survival of marine bacteria during starvation. PLoS Biol. 2010;8:e1000358. [PMC free article] [PubMed]
  • Guindon S, Delsuc F, Dufayard JF, Gascuel O. Estimating maximum likelihood phylogenies with PhyML. Methods Mol Biol. 2009;537:113–137. [PubMed]
  • Howard EC, Sun S, Biers EJ, Moran MA. Abundant and diverse bacteria involved in DMSP degradation in marine surface waters. Environ Microbiol. 2008;10:2397–2410. [PubMed]
  • Huson DH, Richter DC, Rausch C, Dezulian T, Franz M, Rupp R. Dendroscope: an interactive viewer for large phylogenetic trees. BMC Bioinformatics. 2007;8:460. [PMC free article] [PubMed]
  • Kemena C, Notredame C. Upcoming challenges for multiple sequence alignment methods in the high-throughput era. Bioinformatics. 2009;25:2455–2465. [PMC free article] [PubMed]
  • Koh EY, Atamna-Ismaeel N, Martin A, Cowie RO, Beja O, Davy SK, et al. Proteorhodopsin-bearing bacteria in Antarctic sea ice. Appl Environ Microbiol. 2010;76:5918–5925. [PMC free article] [PubMed]
  • Kutschera U. Plant-associated methylobacteria as co-evolved phytosymbionts: a hypothesis. Plant Signal Behav. 2007;2:74–78. [PMC free article] [PubMed]
  • Lanyi JK, Balashov SP. Xanthorhodopsin: a bacteriorhodopsin-like proton pump with a carotenoid antenna. Biochim Biophys Acta. 2008;1777:684–688. [PMC free article] [PubMed]
  • Lindow SE, Brandl MT. Microbiology of the phyllosphere. Appl Environ Microbiol. 2003;69:1875–1883. [PMC free article] [PubMed]
  • Maddison WP, Maddison DR. Mesquite: a modular system for evolutionary analysis. Version 2.73. 2010. [WWW document]. URL
  • Man D, Wang W, Sabehi G, Aravind L, Post AF, Massana R, et al. Diversification and spectral tuning in marine proteorhodopsins. EMBO J. 2003;22:1725–1731. [PubMed]
  • Meyer F, Paarmann D, D’Souza M, Olson R, Glass EM, Kubal M, et al. The metagenomics RAST server – a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics. 2008;9:386. [PMC free article] [PubMed]
  • Muller J, Creevey CJ, Thompson JD, Arendt D, Bork P. AQUA: automated quality improvement for multiple sequence alignments. Bioinformatics. 2010;26:263–265. [PubMed]
  • Oesterhelt D, Stoeckenius W. Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nat New Biol. 1971;233:149–152. [PubMed]
  • Oh HM, Kwon KK, Kang I, Kang SG, Lee JH, Kim SJ, Cho JC. Complete genome sequence of ‘Candidatus Puniceispirillum marinum’ IMCC1322, a representative of the SAR116 clade in the Alphaproteobacteria. J Bacteriol. 2010;192:3240–3241. [PMC free article] [PubMed]
  • Price MN, Dehal PS, Arkin AP. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol. 2009;26:1641–1650. [PMC free article] [PubMed]
  • Qvit-Raz N, Jurkevitch E, Belkin S. Drop-size soda lakes: transient microbial habitats on a salt-secreting desert tree. Genetics. 2008;178:1615–1622. [PubMed]
  • Rusch DB, Halpern AL, Heidelberg KB, Sutton G, Williamson SJ, Yooseph S, et al. The Sorcerer II Global Ocean Sampling expedition: I, The northwest Atlantic through the eastern tropical Pacific. PLoS Biol. 2007;5:e77. [PMC free article] [PubMed]
  • Sabehi G, Loy A, Jung KH, Partha R, Spudich JL, Isaacson T, et al. New insights into metabolic properties of marine bacteria encoding proteorhodopsins. PLoS Biol. 2005;3:e173. [PMC free article] [PubMed]
  • Sabehi G, Kirkup BC, Rosenberg M, Stambler N, Polz MF, Béjà O. Adaptation and spectral tuning in divergent marine proteorhodopsins from the eastern Mediterranean and the Sargasso Seas. ISME J. 2007;1:48–55. [PubMed]
  • Sharma AK, Zhaxybayeva O, Papke RT, Doolittle WF. Actinorhodopsins: proteorhodopsin-like gene sequences found predominantly in non-marine environments. Environ Microbiol. 2008;10:1039–1056. [PubMed]
  • Sharma AK, Sommerfeld K, Bullerjahn GS, Matteson AR, Wilhelm SW, Jezbera J, et al. Actinorhodopsin genes discovered in diverse freshwater habitats and among cultivated freshwater Actinobacteria. ISME J. 2009;3:726–737. [PubMed]
  • Spudich JL. The multitalented microbial sensory rhodopsins. Trends Microbiol. 2006;14:480–487. [PubMed]
  • de la Torre JR, Christianson L, Béjà O, Suzuki MT, Karl D, Heidelberg JF, DeLong EF. Proteorhodopsin genes are widely distributed among divergent bacterial taxa. Proc Natl Acad Sci USA. 2003;100:12830–12835. [PubMed]
  • Yutin N, Suzuki MT, Teeling H, Weber M, Venter JC, Rusch D, Béjà O. Assessing diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of the Atlantic and Pacific Oceans using the Global Ocean Sampling expedition metagenomes. Environ Microbiol. 2007;9:1464–1475. [PubMed]