Astrobiology

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Spectral signatures of photosynthesis I: Review of Earth organisms

Journal: Astrobiology

ManuscriptFor ID: AST-2006-0105 Peer Review

Manuscript Type: Research Articles (Papers)

Date Submitted by the 22-Nov-2006 Author:

Complete List of Authors: Kiang, Nancy; NASA Goddard Insitute for Space Studies; CalTech, Infrared Processing and Analysis Center (IPAC) Siefert, Janet; Rice University, Dept. of Statistics Govindjee, Govindjee; University of Illinois at Urbana-Champaign, Departments of Plant Biology and Biochemistry Blankenship, Robert; Washington University, Department of Biology and Chemistry Meadows, Victoria; California Institute of Technology, Spitzer Science Center

Anoxygenic Photosynthesis, Oxygenic Photosynthesis, Astrobiology, Keyword: Spectroscopic Biosignatures, Red Edge

Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 1 of 69 Astrobiology

1 2 3 Short title: 4 5 Spectral signatures of photosynthesis: Earth review 6 7 Full title: 8 Spectral signatures of photosynthesis I: Review of Earth organisms 9 10 *Nancy Y. Kiang 1,6 , Janet Siefert 2,6 , Govindjee 3, Robert E. Blankenship, 4 Victoria S. 11 2,6 12 Meadows 13 14 1NASA God dard Institute for Space Studies, U.S.A. 15 2Dept. of Statistics, Rice University 16 3Departments of Plant Biology and Biochemistry, University of Illinois at Urbana - 17 18 Champaign, U.S.A. 4 For Peer Review 19 Department of Biology and Chemistry, Washington University, U.S.A. 20 5Spitzer Science Center, California Institute of Technology, USA 21 6NASA Astrobiology Institute 22 23 24 *To whom correspondence should be addressed: Nancy Y. Kiang, NASA Goddard 25 Institute for Space Studies, New York, NY 10025, fax1: (626) 568 -0673, fax2: (212) 26 678 -5552, t el1: (626) 395 -1815, tel2: (212) 678 -5587, cell: (949) 439 -3416, email: 27 [email protected] 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 2 of 69

1 DRAFT , Kiang, Astrobiology 2 3 4 5 Abstract 6 7 8 9 Why do plants reflect in the green and have a “red edge” in the red, and should extrasolar 10 11 photosynthesis be the same? We provide: 1) a brief review of how photosynthesis works; 2) an 12 13 overview of the diversity of photosynthetic organisms, their light harvesting systems, and 14 15 16 environmental ranges; 3) a synthesis of photosynthetic surface spectral signatures; 4) 17 18 evolutionary rationalesFor for photosynthetic Peer surface Review reflectance spectra with regard to utilization of 19 20 photon energy and the planetary light environment. We found the “NIR end” of the red edge to 21 22 trend from blue -shifted to reddest for (in order): snow algae, temperature algae, lichens, mosses, 23 24 aquat ic plants, and finally terrestrial vascular plants. The red edge is weak or sloping in lichens. 25 26 Purple bacteria exhibit possibly a sloping edge in the NIR. More studies are needed on pigment - 27 28 29 protein complexes, membrane composition, and measurements of ba cteria before firm 30 31 conclusions can be drawn about the role of the NIR reflectance. Pigment absorbance features are 32 33 strongly correlated with features of atmospheric spectral transmittance: P680 in PS II with the 34 35 peak surface incident photon flux density a t ~685 nm, just before an oxygen band at 687.5 nm; 36 37 the NIR end of the red edge with water absorbance bands and the oxygen A -band at 761 nm; and 38 39 bacteriochlorophyll reaction center wavelengths with local maxima in atmospheric and water 40 41 42 transmittance spectr a. Given the surface incident photon flux density spectrum and resonance 43 44 transfer in light harvesting, we propose some rules with regard to where photosynthetic pigments 45 46 will peak in absorbance: a) the wavelength of peak incident photon flux; b) the longes t available 47 48 wavelength for core antenna or reaction center pigments; and c) the shortest wavelengths within 49 50 an atmospheric window for accessory pigments. That plants absorb less green light may not be 51 52 an inefficient legacy of evolutionary history, but ma y actually satisfy the above criteria. 53 54 55 56 57 58 59 60 2 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 3 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 4 5 1. Introduction 6 7 8 9 10 The utilization of the Sun’s light energy by photosynthetic organisms provides the 11 12 foundation for virtually all life on Earth, with the annual amount of carbon fixed from 13 14 CO 2 into organic form by la nd plants and by ocean phytoplankton being each 15 16 17 approximately ~ 45 -60 Pg -C/yr (Cramer, et al., 1999, 2001), or 6 -8% of the atmospheric 18 For Peer Review 19 carbon content (Reeburgh, 1997). The selective utilization of light energy results in two 20 21 well -known spectral signatures exhibited in land plants: the “green bump,” due to lower 22 23 24 absorbance of green light by chlorophyll; and the “red edge,” characterized by 25 26 absorbance in the red by chlorophyll strongly contrasting with reflectance in the near - 27 28 infrared due to refraction betwe en leaf mesophyll cell walls and air spaces in the leaf. 29 30 31 The red edge is so different spectrally from other matter that it has been measured by 32 33 satellites to identify vegetation cover (Tucker, 1976; Grant, 1987; Sagan, et al., 1993) and 34 35 36 estimate plant pr oductivity (Potter, et al., 1993). 37 38 We seek to address and extend the age -old question: Why are plants green? More 39 40 precisely, what is the functional role of different features of a photosynthetic organism’s 41 42 43 reflectance spectrum? Although it is fairly wel l understood how pigments absorb and 44 45 how cells scatter light, it is not yet settled as to why photosynthetic pigments absorb at 46 47 those particular wavelengths. Finally, how ubiquitous is the near -infrared (NIR) 48 49 50 reflectance among photosynthetic organisms, an d how does it serve the organism ? 51 52 Photosynthetic spectral reflectance signatures are a result of both molecular 53 54 constraints on biochemical processes and environmental pressures for adaptation. In this 55 56 57 58 59 60 3 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 4 of 69

1 DRAFT , Kiang, Astrobiology 2 3 review, we attempt to synthesize spectral characteri stics across the full range of Earth’s 4 5 6 photosynthesizers, covering the following: 7 8 9 10 11 1) a brief review of how photosynthesis works; 12 13 2) an overview of the diversity of photosynthetic organisms, their light harvesting 14 15 systems, and environmental ranges; 16 17 18 3) a sy nthesis of photosyntheticFor Peer surface spectral Review signatures; 19 20 4) an exploration of evolutionary rationales for photosynthetic surface reflectance 21 22 spectra, including the Earth’s chemical history, energy requirements for conversion of 23 24 25 photon energy to chemical ener gy, and the planetary light environment. 26 27 28 29 We conclude with some hypotheses about why photosynthetic pigments favor their 30 31 32 particular wavelengths, and whether alternative whole organism reflectance spectra could 33 34 be possible. As this review is motivated by speculation about photosynthesis on Earth - 35 36 37 like planets in other solar systems, much of the discussion is placed in this context and 38 39 geared toward the diverse multi -disciplinary astrobiology audience. This review should 40 41 also be useful to specialists in Ear th remote sensing, photosynthesis, and plant 42 43 44 physiology. 45 46 47 48 49 2. Background: Basic processes of photosynthesis, inputs and outputs 50 51 52 53 Photosynthesis efficiently converts light energy to electrochemical energy for 54 55 56 oxidation -reduction (“redox”) reactions. The exci tation of light harvesting pigments by a 57 58 59 60 4 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 5 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 photon of light causes an electron to be transferred along biochemical pathways that lead 4 5 6 to the reduction of CO 2. The electron is replaced by one extracted from the reductant. 7 8 The basic stoichiometry of photosynth esis is: 9 10 11 12 13 CO 2 + 2H2 A + hν → ()CH 2O + H2O + 2A ( 1 ) 14 pigments carbohydrate 15 16 17 18 For Peer Review 19 where H 2A is a reducing substrate such as H 2O or H 2S, and h ν is the energy per photon, 20 21 22 where h is Planck’s constant, and ν is the frequency of the photon or the speed of light 23 24 divided by the photon wavelength. 25 26 27 When the reductant is water, then we have oxygenic photosynthesis: 28 29 30 31 c w c + w − c c w 32 6CO 2 +12 H2O + hν → 6CO 2 + 24 H + 6O2 + 24 e →()C6H12 O6 + 6H2O + 6O2 ( 2 ) 33 glu cos e 34 35 36 37 38 where the superscripts c and w denote the oxygen from carbon dioxide versus that from 39 40 water. Four photons are req uired for each O 2 evolved (one photon for each bond in two 41 42 water molecules), and four photons are needed to reduce two molecules of the coenzyme 43 44 45 NADP+ eventually to reduce one CO 2. Thus, a minimum of 8 photons total are required 46 47 both to evolve one O 2 and to fix carbon from one CO 2. A few cycles are required to 48 49 50 obtain the six carbons to make the 6 -carbon sugar, glucose. More than 8 photons are 51 52 generally required (experiments have shown up to about 12 photons; Govindjee, 1999) 53 54 because some are unsuccessful , and some in addition are used in the processes of cyclic 55 56 57 photophosphorylation (occurs in Photosystem I generation of ATP, described later; Joliot 58 59 60 5 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 6 of 69

1 DRAFT , Kiang, Astrobiology 2 3 and Joliot, 2002; Munekage, et al., 2004) and nitrogen assimilation (Foyer and Noctor, 4 5 6 2002). The mechanism s by which these processes are achieved are highly complex, and 7 8 the reader is referred to details in textbooks and recent findings (Voet, et al., 1999; Ke, 9 10 11 2001; Green and Parson, 2004; Ferreira, et al., 2004; Wydrzynski and Satoh, 2005). 12 2+ 13 Photosynthesi s may use reductants other than water, such as H 2S, H 2, and Fe in 14 15 anoxygenic photosynthesis. When the reductant is H 2S, then elemental sulfur is produced 16 17 18 instead of oxygen, Forand that sulfur Peer may be further Review oxidized to sulfate (Van Gemerden and 19 20 Mas, 1995): 21 22 23 24 25 CO 2 + 2H2S + hν → ()CH 2O + H2O + 2S ( 3a ) pigments 26 carbohydrate 27 28 29 3CO 2 + 2S + 5H2O + hν → 3()CH 2O + 2H2SO 4 ( 3b ) pigments 30 carbohydrate 31 32 2+ 33 When the reductant is H 2 (Vignais, et al., 1985) or Fe (Jiao, et al., 2005; Ehrenreich 34 35 and Widdel, 1994), the reactions are: 36 37 38 39 40 CO 2 + 2H2 + hν → (CH 2O)+ H2O ( 4 ) 41 42 CO +4 Fe 2+ +11 H O + hν → (CH O)+ 4 Fe (OH ) +8H + ( 5 ) 43 2 2 2 3 44 45 46 - 47 (In Equation 5, actually HCO 3 and not CO 2 is directly used, though it could come from 48 49 2+ 50 CO 2 dissolved in water or from dissolution of some other carbonate source; the Fe 51 52 could be from dissolution of, e.g. FeCO 3). 53 54 The quantum requirement in all these cases is also 8 -12 photons per carbon fixed. 55 56 57 In summary, the inputs to photosynthesis are light energy, a carbon source, and a 58 59 60 6 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 7 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 reductant. The direct products are carbohydrates and ca n be oxygen, elemental sulfur, 4 5 6 water, and other oxidized forms of the reductant. Soil nutrients are also necessary inputs, 7 8 described in more detail below . 9 10 11 12 13 14 15 16 17 18 3. Range of photosyntheticFor Peer organisms, Review habitats, pigments, metabolisms, 19 20 and environmental limits 21 22 23 24 25 Photosynthetic organisms are adapted to occupy different niches according to 26 27 both physical (light, temperature, moisture) and chemical resource (electron donors, 28 29 30 carbon sources, nutrients) requirements. In addition, their environmental ranges may be 31 32 contr olled by the presence of competing or grazing organisms. The range of 33 34 photosynthetic organisms is shown in Table 1, which lists the approximate time of their 35 36 37 appearance on Earth, the significant features that distinguish them metabolically and 38 39 spectrally, their relative abundance and productivity over the Earth, habitats, their 40 41 pigments types, reaction center types, electron donors, growth mode and growth form, 42 43 44 carbon sources, and metabolic products. 45 46 Emergence of photosynthetic organisms on Earth. Photos ynthesis arose fairly 47 48 49 early in the history of the Earth. One theory about the origin of photosynthesis is that it 50 51 began as a fortuitous adaptation of primitive pigments for infrared thermotaxis of 52 53 chemolithotrophic bacteria in hydrothermal ocean vents (ea rly Archean, possibly 3.8 Ga 54 55 56 ago; Nisbet, et al., 1995). Thus, less dependent on the heat of the hydrothermal vents, the 57 58 59 60 7 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 8 of 69

1 DRAFT , Kiang, Astrobiology 2 3 habitats of these organisms gradually expanded to shallower waters where solar light 4 5 6 could be utilized (Nisbet and Fowler, 1999; Des Marais, 2000). It is also possible that 7 8 photosynthesis arose first in shallow waters and, before there was oxygenic 9 10 11 photosynthesis to provide an ozone shield, UV screening proteins led to the transfer of 12 13 excitation energy to the porphyrin (Mulkidjanian an d Junge, 1997). The evolution of 14 15 ocean chemistry could have played a major role in the evolution of chlorophyll 16 17 18 (Mauzerall, 1976; ForDasgupta, etPeer al., 2004; Dismukes, Review 2001; Blankenship and Hartman, 19 20 1998). Blankenship and Hartman (1998) proposed that hydroge n peroxide, H 2O2, could 21 22 have been a transitional electron donor on the oxygen -poor early Earth. Liang, et al. 23 24 25 (2006) posited that the necessary high H 2O2 in the oceans could have occurred after a 26 27 “Snowball Earth” event (low -latitude glaciation), around 2. 3 Ga, due to storage of H 2O2 28 29 in ice and its subsequent release into the oceans upon de -glaciation. Protocyanobacteria 30 31 + 32 early in the Archean may have utilized Fe(OH) as a reductant (Olson, 2006). Dismukes, 33 34 et al. (2001) proposed that bicarbonate could hav e been a transitional reductant before 35 36 37 water in the Archean ocean and reacted with manganese (II) to form Mn -bicarbonate 38 39 clusters as precursors to the (Mn) 4 core of the oxygen evolving complex; the redox 40 41 potentials are such that green sulfur bacteria woul d have been the early hosts of these 42 43 44 clusters prior to the evolution of cyanobacteria. 45 46 The fossil record seems to support that the early photosynthesizers were 47 48 anoxygenic purple bacteria and green sulfur bacteria that used reducing substrates other 49 50 51 than water, such as H 2 and H 2S (Olson, 2006). Anoxic environments allowed these 52 53 organisms to thrive, since oxygen is damaging to bacteriochlorophylls. By the mid - to 54 55 late -Archean (3.5 -3.6 Ga ago) or as late as 2.3 Ga ago in the Early Proterozoic, oxygenic 56 57 58 59 60 8 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 9 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 cyan obacterial mats had formed in the shallower waters. Endosymbiosis of 4 5 6 cyanobacteria and early animal protists gave rise to the plastids of algae, these plastids 7 8 being the photosynthesizing organelle (Larkum, et al., 2003). The algae were, thus, the 9 10 11 first eukaryotic photosynthesizers and occur in both single -cell and multi -cellular forms 12 13 (e.g. kelp). Red algae appeared around 1.2 Ga ago. Eukaryotic green algae did not 14 15 appear until as late as 750 Myr ago. These photosynthetic organisms were protected 16 17 18 unde r water from UVFor radiation Peer until O 2 and, hence,Review O 3 built up in the atmosphere. With 19 20 the rise of atmospheric O 2, the anoxygenic ancestors lost their competitive advantage to 21 22 organisms that could withstand and respire O 2 (or so one may interpret). Land plan ts are 23 24 25 believed to be descended from a single branch of the green algae (Larkum, et al., 2003). 26 27 The first land plants, with the non -vascular Bryophyta (mosses and liverworts) being the 28 29 earliest, did not occur until 460 Myr ago, after which the abundance o f plant life 30 31 32 exploded, reaching a peak of productivity during the Carboniferous period 354 Myr ago 33 34 (Carroll, 2001). Further complexity continued to arise through the emergence of 35 36 37 flowering plants (144 Myr ago), and more water -efficient photosynthetic path ways came 38 39 about through anatomical and enzymatic changes . In crassulacean acid metabolism 40 41 (CAM) photosynthesis, which arose 70 -55 Myr ago, CO 2 is stored in an intermediate at 42 43 44 night ; d uring the day, stomates remain closed to prevent water loss, and the int ermediate 45 46 is broken down for CO 2 to enter the Calvin cycle. In C4 photosynthesis, which arose 20 - 47 48 35 Myr ago, CO is similarly concentrated in an intermediate that is transported to an 49 2 50 51 internal bundle of cells that allow stomates to draw in atmospheric CO 2 through a smaller 52 53 aperture (Sage, 2001). 54 55 56 57 58 59 60 9 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 10 of 69

1 DRAFT , Kiang, Astrobiology 2 3 So it seems natural for organisms to evolve to capture stellar energy, and Earth’s 4 5 6 example shows that, they continue to get better at it , especially once they emerge from 7 8 the water. Earlier organisms are more div erse in their photosystems, while later 9 10 11 organisms are more complex in their morphological properties. Note that not all 12 13 photosynthesizers are autotrophs (fix CO 2), but many of the bacteria are heterotrophs that 14 15 utilize organic carbon, though some may use both inorganic and organic carbon. The 16 17 18 halobacteria do notFor perform actual Peer photosynthesis, Review in that no electron transfer is 19 20 performed, but their pigment bacteriorhodopsin drives a proton pump for heterotrophic 21 22 assimilation of organic carbon. Of greatest in terest to us are those photosynthesizers that 23 24 25 are autotrophic and can fix CO 2 directly, as these are the primary producers and the 26 27 foundation for life on Earth. We survey next their photosystems and environmental 28 29 constraints. 30 31 32 33 34 Light harvesting. Photosynt hetic organisms have intricate photosystems that coordinate 35 36 37 1) the spectral selection of light energy and 2) the abstraction of electrons from electron 38 39 donors. These photosystems are composed of three main components. The 1) peripheral 40 41 or outer antenna co mplex transfers light energy to 2) the core or inner antenna . These 42 43 44 two together are known as the light -harvesting complex (LHC). The core antenna is also 45 46 an integral part of the third component, 3) the reaction center (RC) complex, where light 47 48 energy is finally converted to chemical energy in charge separation (e.g. with H 2O, other 49 50 51 electron donors, enzymes) (Ke, 2001). 52 53 The photon utilized must be of sufficient energy to generate a voltage potential 54 55 difference that is great enough to oxidize the reductan t as well as afford the electron 56 57 58 59 60 10 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 11 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 transfers for reduction of the relevant intermediates (and some enzymes). In plants and 4 5 6 all oxygenic photosynthesis, there are two stages of light utilization: 1) in the extraction 7 8 of electrons from water (using Photosyst em II or PS II, peaking in absorbance at 680 nm) 9 10 + 11 to regenerate the next step; and 2) for the reduction of the electron carrier NADP (using 12 13 Photosystem I or PS I, peaking in absorbance at 700 nm), which is then used in the 14 15 Calvin -Benson Cycle and for the s ynthesis of ATP. This two -step sequence is known as 16 17 18 the “Z -scheme” forFor the zig -zag Peer in redox potential Review at each step (first proposed by Hill and 19 20 Bendall, 1960; first proven by Duysens, et al., 1961; Blankenship and Prince, 1985). 21 22 The redox potential is th e Gibbs free energy change of a reaction (calculated with the 23 24 25 Nernst equation), which may be expressed in volts and is the propensity of an oxidation - 26 27 reduction reaction to proceed spontaneously in one direction or the reverse; the 28 29 convention is that hydrog en has a redox potential of zero (at equilibrium conditions) and 30 31 32 electrons move spontaneously in the direction of higher (toward positive) potentials. 33 34 These thermodynamics determine how much energy may be stored as product, while the 35 36 37 kinetics of electron transfer affect light harvesting and the quantum yield. All oxygenic 38 39 photosynthesizers utilize both PS I and PS II, with water as the reductant. Anoxygenic 40 41 photosynthesizers utilize only a single photosystem that uses other electron donors, for 42 43 44 which equ ations were given earlier. Figure 1 shows the reaction center midpoint redox 45 46 potentials (when concentrations of reductants and oxidants are the same) and excited 47 48 potentials for the Z -scheme of oxygenic photosynthesis, as well as the electron transport 49 50 51 pat hways for different classes of bacteria. The Type I and Type II reaction center 52 53 categories distinguish the types of electron acceptors used. Only oxygenic 54 55 56 57 58 59 60 11 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 12 of 69

1 DRAFT , Kiang, Astrobiology 2 3 photosynthesis is known to utilize two reaction centers in sequence. For a more detailed 4 5 6 overview, the reader is referred to Blankenship (2002). 7 8 Photosynthetic pigments are categorized into three chemical groups or 9 10 11 “chromophores”: the chlorophylls, carotenoids, and phycobilins. The chlorophylls are 12 13 the pigments of the reaction centers and also occur i n the core antennae and light 14 15 harvesting antennae. Specialized chlorophylls at the RCs serve to “trap” the excitation 16 17 18 energy and convertFor the electronic Peer energy to chemicalReview energy through charge separation. 19 20 The funneling of energy from the LHCs in all euka ryotes is achieved through a rather 21 22 remarkable process known as resonance excitation transfer (also known as the Förster 23 24 25 mechanism; review: Clegg, 2004), in which a pigment is excited by light at a particular 26 27 wavelength and the subsequent de -excitation of the pigment, rather than resulting in a 28 29 loss of energy to heat or fluorescence, leads to the excitation of another pigment whose 30 31 32 energy level overlaps. A series of such excitations and de -excitations creates an “energy 33 34 cascade” toward longer wavelengths. In all oxygenic eukaryotes, Chl a occurs in the core 35 36 37 antenna and acts as the primary donor in the reaction center; H 2O is the electron donor. 38 39 Other chlorophylls (Chl b/c/d) provide light harvesting roles, but very recently Chl d, 40 41 which was discovered in cyanobacteria (Miyashita, et al., 1996; Miller, et al., 2005), may 42 43 44 replace Chl a in the reaction centers in some cyanobacteria that live in environments with 45 46 little visible light (Larkum and Kühl, 2005; Chen, et al., 2005;). Chl d has its major peak 47 48 abs orbance in the NIR at ~720 nm (Manning and Strain, 1943; Larkum and Kühl, 2005), 49 50 51 and thus, oxygenic photosynthesis is being performed in the NIR! In non -oxygenic 52 53 bacteria, bacteriochlorophylls play the role of primary donor, with a great variety having 54 55 56 57 58 59 60 12 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 13 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 distinct absorption spectra; a variety of electron donors are possible, including H S, H , 4 2 2 5 6 Fe, sulfate, thiosulfate, sulfite, and organic carbon. 7 8 The other pigments as well as additonal chlorophylls (particularly Chl b in plants 9 10 11 and green algae, and Chl c in diatoms and brown algae) in the light harvesting complexes 12 13 (LHCs) act as “accessory” pigments to help obtain light energy, the carotenoids in the 14 15 blue and green, and the phycobilins (cyanobacteria and red algae) in the green and 16 17 18 yellow. The carotenoidsFor in addition Peer serve to protectReview chlorophyll against photo -oxidative 19 20 damage in conditions of high light, high temperature, O 2, and the presence of certain 21 22 pigments (Nobel, 1999). Oxygen would be toxic to photosynthetic organisms without the 23 24 25 presence of carot enoids. 26 27 The absorbance spectra of all pigments are influenced by the protein complexes in 28 29 which they are bound, such that there can be variations in the same type of pigment and 30 31 32 the peak absorbances in vivo tend to be broadened and shifted compared to t hose of the 33 34 pure pigments extracted in solution (Vasil’ev and Bruce, 2006). For example, Chl a in 35 36 37 PS I peaks at 700 nm, whereas in PS II it peaks at 680 nm. We compare in vivo pigment 38 39 spectra (in intact membranes) below. 40 41 Figure 2a shows the in vivo abso rbance spectra of the major pigments found in 42 43 44 plants and algae (Chl a, Chl b, carotenoids; phycobilins exist only in certain algae and 45 46 cyanobacteria), as well as the spectrum of Chl a fluorescence (Papageorgiou and 47 48 Govindjee, 2004), together with the inci dent solar radiation spectrum at the top of the 49 50 51 Earth’s atmosphere and at the surface of the Earth after atmospheric filtering. Sources 52 53 and measurement methods for the pigment spectra are summarized in Appendix A1. The 54 55 Chl a fluorescence spectrum effective ly identifies the upper wavelength limit to 56 57 58 59 60 13 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 14 of 69

1 DRAFT , Kiang, Astrobiology 2 3 absorbance by chlorophyll. The photon flux densities are from models, as detailed in the 4 5 6 figure caption; in addition, measurements from buoys at Hawaii (Dennis Clark, NOAA) 7 8 are plotted to show how varying atmosp heric optical depth can affect the incident light 9 10 11 spectrum. The atmosphere performs spectral filtering of radiation, with Mie and 12 13 Rayleigh scattering of light in the bluer wavelengths, and with clear bands of absorption 14 15 by gases, most importantly O 3, O 2, H2O vapor, and CO 2, as indicated in the figure. Plant 16 17 18 and algae pigmentsFor clearly must Peer have evolved Review to have their absorbance peaks in 19 20 atmospheric transmittance windows for radiation and are confined to the visible range, 21 22 which is commonly considered to be “photosynthetically active radiation” (PAR), 400 - 23 24 25 700 nm (the tail of the absorbed range extends as far as 730 nm). 26 27 Figure 2b shows the in vivo absorbance spectra of the bacteriochlorophylls with 28 29 associated carotenoids in intact membranes (sources and me asurement method 30 31 32 summarized in Appendix A1). Above these are plotted the transmitted radiation 33 34 spectrum through 5 cm of pure water as well as through 10 cm of water containing algae 35 36 37 (water absorption coefficient from Segelstein, 1981, Sogandares and Fry, 1997, and Kou, 38 39 et al., 1993; algae absorption coefficient from kelp, see Appendix A3 for explanation of 40 41 calculation). Water is highly transmitting in the visible and highly absorbing in the NIR, 42 43 44 as can be seen from the spectrum for pure water at 5 cm dept h. A small transmittance 45 46 window exists, however, in the NIR, peaking at 1073 nm. Algae and cyanobacteria in the 47 48 upper layers of water may strongly attenuate visible light, such that only radiation above 49 50 51 about 700 nm may be transmitted to depth and attenu ated again by water above 900 nm. 52 53 The cyanobacterium, Acaryochloris marina , which uses Chl d at ~715 -720 nm instead of 54 55 Chl a, may be adapted to receive this remaining longer wavelength filtered through 56 57 58 59 60 14 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 15 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 overlying organisms. The bacteriochlorophylls common ly absorb in the range 700 -900 4 5 6 nm. In an extreme case, BChl b in the purple bacteria Blastochloris viridis (formerly 7 8 Rhodopseudomonas viridis ) can absorb to wavelengths as long as 1013 -1025 nm (Scheer, 9 10 11 2003; Trissl, 1993). B. viridis inhabits murky, ano xic sediments where little visible light 12 13 penetrates. Its exact peak absorbance is sensitive to temperature, and the range of 14 15 measurements on lab cultures may, perhaps, not be exactly the same as in the bacterium’s 16 17 18 native environment.For The local Peer peak in wa ter transmittanceReview at 1073 nm leaves room for 19 20 speculation that either B. viridis has the capability to harvest light at even longer 21 22 wavelengths, or the wavelength of peak absorbance is limited by exciton transfer kinetics 23 24 25 to the reaction center or thermodyn amic constraints. 26 27 In addition to the bacteriochlorophylls, phototrophic bacteria will also utilize 28 29 carotenoids, with the ratio between bacteriochlorophyll and carotenoids varying with 30 31 32 light quality. In Figure 2b, the absorbance peaks in the blue wavele ngth range are due to 33 34 carotenoids. Purple and green sulfur bacteria pigments thus absorb in transmittance 35 36 37 windows under water. Overall, the full range of pigments has been observed to absorb 38 39 light in the wavelength ranges 330 -900 nm and 1000 -1100 nm (Sch eer, 2003). Oxygenic 40 41 photosynthesis on Earth is limited to photosystems that operate at 400 -730 nm, but 42 43 44 anoxygenic photosynthesis occurs at wavelengths as long as 1015 -1020 nm (Trissl, 1993; 45 46 Scheer, et al, 2003). 47 48 To summarize (see Table 1), phototrophic bacteria utilize bacteriochlorophylls in 49 50 51 their reaction centers and are the most diverse in their growth modes, many being 52 53 heterotrophs and some using sulfide, iron, or hydrogen as their electron donors (Eraso 54 55 and Kaplan, 2001; Ehrenreich and Widdel, 1994) . Among the phototrophic bacteria, only 56 57 58 59 60 15 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 16 of 69

1 DRAFT , Kiang, Astrobiology 2 3 cyanobacteria (formerly known as blue -green algae) are oxygenic. The diversity of 4 5 6 pigments of cyanobacteria includes Chl a/b/c/d, phycobilins, and carotenoids, which 7 8 allows them a wide range of colors and distribut ion nearly everywhere on Earth, from 9 10 11 aquatic environments to desert salt crusts. Where cyanobacteria or algae coexist with 12 13 fungi in lichens, they can support a level of productivity comparable to vascular plants. 14 15 All algae utilize Chl a and carotenoids; in addition, green algae use Chl b. Several other 16 17 18 algae (e.g diatoms Forand brown algae)Peer use different Review forms of Chl c, and red algae utilize 19 20 phycobilins instead of Chl b or Chl c. Plants utilize only Chl a, Chl b, and carotenoids 21 22 but have developed more com plex mechanisms to acquire CO 2 and retain water. All 23 24 25 photosynthetic organisms have carotenoids. 26 27 Because the photosystems are tied to the type of electron donor, the 28 29 photosynthesizer is therefore necessarily restricted to particular resource environment 30 31 32 niches. Purple and green sulfur bacteria are restricted to aquatic environments where 33 34 sulfur (sulfide, sulfate, sulfite, thiosulfate, H 2S) is available as an electron donor, and 35 36 37 some of these bacteria require anoxic environments. Cyanobacteria and other 38 39 photosynthetic bacteria are also known to form mutualistic communities in microbial 40 41 mats in benthic (Decker, et al., 2005) and freshwater aquatic environments (Wiggli, et al., 42 43 44 1999), where distinct layering demarcates both light and chemical niches. Exudat es and 45 46 organic carbon from one layer provide the electron source or food source for another, and 47 48 the lower, anoxic layers utilize sulfide or H S for the electron donor. The upper layers of 49 2 50 51 cyanobacteria and algae absorb visible light, such that the lower layers of sulfur bacteria 52 53 utilize the NIR radiation that transmits through. 54 55 56 57 58 59 60 16 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 17 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 Next we will comment briefly on the other environmental constraints on 4 5 6 photosynthetic organisms, as these can affect also their spectral properties. 7 8 Climate – water and tempe rature. Water and temperature are the primary 9 10 11 constraints on the distribution, spatially and temporally, of different organisms. For 12 13 plants, the reader is referred to well -known global surveys of the climate limits of, for 14 15 example, grasslands, broadleaf temperate deciduous forests, evergreen needleleaf forests, 16 17 18 boreal rainforest, toFor name some Peer possible categories Review of many (Holdridge, 1967; 19 20 Whittaker, 1975; Woodward, 1987; Larcher, 1995). These large -scale classifications 21 22 show clear correlations between climate and plant form and mixtures of plant 23 24 25 communities, such as broadleaf trees in temperate and tropical zones versus needleleaf 26 27 trees in colder climates. The resulting difference in spectral signatures can be 28 29 distinguished by satellites (Tucker, et al ., 1985; Defries and Townshend, 1994). Mosses 30 31 32 and liverworts, which lack vascular structure, require very moist and often shaded 33 34 environments, and do not form large structures. Lichens (symbioses between fungi and 35 36 37 algae or cyanobacteria) are limited to e nvironments with moist air but can be highly 38 39 productive. They are often the first colonizers on rock substrate and can be the dominant 40 41 source of net primary productivity and a source of food for animals in some land 42 43 44 ecosystems (Ager, et al, 1987; Rees, et al., 2004), in some cases accounting for 70% of 45 46 the land cover (Solheim, et al., 2000). The extreme temperature limits of photosynthetic 47 48 organisms range as low as -15.7 ºC, the survival limit for arctic snow algae and arctic ice 49 50 51 shelf cyanobacteria (but they require liquid water for growth) (Gorton, et al., 2001; 52 53 Mueller, et al., 2005) and as high as ~75ºC for bacteria in hot springs (Miller, et al., 54 55 1998). Meanwhile, the temperature limits for Earth life in general can be as low as -20 56 57 58 59 60 17 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 18 of 69

1 DRAFT , Kiang, Astrobiology 2 3 °C (possibly as low as -196 °C for some methanogenic bacteria) (Junge, et al., 2006) and 4 5 6 possibly as high as 120 °C (Kashefi and Lovley, 2003). 7 8 Extreme environments. Photosynthetic bacteria and some algae tend to inhabit 9 10 11 what on Earth are considered extreme or stress ful conditions, such as low light (e.g. B. 12 13 viridis ), desert (Karnieli, et al., 1999), snow and ice (algae, Gorton, et al., 2001; 14 15 cyanobacteria, Mueller, et al., 2005), saline (Decker, et al., 2005), low pH (pH of 0, red 16 17 18 algae, Schleper, et Foral., 1995; archaPeerea at pH 0, ReviewEdwards, et al., 2000), high pH (pH of 10, 19 20 cyanobacteria, Finlay, et al, 1987), and anoxic environments. 21 22 Light quantity. Some organisms may function better in low or high light 23 24 25 environments. The lower light limit is determined by the balance between photosynthesis 26 27 and respiration, the cut -off between survival and death (the “light compensation point”), 28 29 while the upper limit is determined by other resource limits (water, nutrients, carbon 30 31 32 source) and the ability to protect against photo -oxida tion (damage to chlorophyll due to 33 34 excited states of O 2 in high light). Rather than quantifying light in terms of energy, we 35 36 37 express it here in photons (or moles of photons) because photosynthesis depends on 38 39 photon flux (at particular wavelengths) rather than energy flux. The lowest observed 40 41 light compensation points are ~ 3 µmol -photons m -2 s -1 (0.7 W/m 2, 1.8 x 10 18 photons m -2 42 43 -1 -2 -1 44 s ) of PAR for green plants (Nobel, 1999) and ~ 0.01 µmol -photons m s red macro - 45 46 algae (6 x 10 15 photons m -2 s -1, Littler, e t al., 1986). Overmann, et al. (1992) observed a 47 48 49 brown sulfur bacterium, living at ~80 m depth in the Black Sea, that is adapted to an 50 51 available irradiance of 0.003 -0.01 µmol -photons m -2 s -1 (1.8 -6.0 x 10 15 photons m -2 s -1) 52 53 54 (additional characterization by Manske, et al., 2005). The theoretical unicellular light 55 56 limit has been estimated by Raven (1984) to be ~ 0.1 µmol -photons m -2 s -1 (6 x 10 16 57 58 59 60 18 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 19 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 photons m -2 s -1), and as this is higher than that observed, additional efficiency strategies 4 5 6 for survival at low light must be more a possibility than current understanding allows. 7 8 Recently, Beatty, et al. (2005) discovered a green sulfur bacterium that utilizes 9 10 11 geothermal light at a hydrothermal vent, where the irradiance close to the vent is 12 13 comparable to that at the 80 m depth of the Black Sea. This opens up the possibilities for 14 15 photosynthesis independent of starlight. However, it is unlikely that these bacteria 16 17 18 evolved under suchFor low light conditions,Peer but weReview think they probably are migrants from 19 20 surface waters . For the upper limit of photon flux density, Wolstencroft and Raven 21 22 (2002) summarized the literature and found a theoretical tolerance for land plants against 23 24 -2 -1 21 -2 -1 25 photodamage at 6 -9 mmol -photon m s (3.6 -5.4 x10 photons m s ) over the PAR 26 27 band, which is well above Earth’s typical flux of 2 mmol photon m -2 s -1 (1.2 x 10 21 28 29 photons m -2 s -1). For Earth -like planets in general, they conjectured a theoretical upper 30 31 32 limit for land organisms to be 10 mmol photon m -2 s -1 (6 x 1021 photons m -2 s -1). 33 34 Since aq uatic organisms are shielded under water, they could exist for even higher 35 36 37 surface photon flux densities. 38 39 Ultraviolet light damage. UV light is damaging to DNA, and exposure to UV at 40 41 levels received at the Earth’s surface generally inhibits photosynthes is and leaf expansion 42 43 44 (Karentz, et al., 1994; Nobel, 1999; Tenini, 2004). Therefore, most of the discussion on 45 46 UV effects on habitability focuses on protection against UV by ozone or 47 48 microenvironments (Cockell and Raven, 2004; Cockell, 1999; Kasting, e t al., 1997); 49 50 51 behaviors such as photomotility to avoid the harmful UV (Bebout and Garcia -Pichel, 52 53 1995); and specialized screening pigments or phenolics that prevent the penetration of 54 55 UV light into the cell (scytonemin in arctic cyanobacteria, Mueller, et al., 2005; 56 57 58 59 60 19 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 20 of 69

1 DRAFT , Kiang, Astrobiology 2 3 phenolics in snow algae, Gorton, et al., 2001; mycosporine -like amino acids in response 4 5 6 to UVB photoreceptors in cyanobacteria, Portwich and Garcia -Pichel, 2000). Such 7 8 protective compounds may have played a role in the early emergence of plant s onto land 9 10 11 (Cooper -Driver, 2001; scytonemin found in ancient rocks by Marilyn Fogel, personal 12 13 communication, NASA Astrobiology Institute conference, 2005). The maximum flux of 14 15 UV at the Earth’s surface is 1.8 -2.8x10 18 photons/m 2/s over the UV -B (280 -315 nm) band 16 17 2 18 at the Equator at noonFor under cloudlessPeer conditions, Review or averages globally 0 -12 kJ/m /day 19 20 (2.1x10 17 photons/m 2/s, converting with the average energy per photon in the UV -B). 21 22 Damage to plants from UV -B radiation has been observed at doses of 15 -16 kJ /m 2/day 23 24 17 2 25 (2.6 -2.8x10 photons/m /s, Kakani, et al., 2003). 26 27 Nutrients. Nutrients, as mentioned earlier, are also limiting resources. The 28 29 nature of the limitation is on level of productivity and competitive advantage, rather than 30 31 32 physiological tolerance. On land, the succession of species is often a function of the 33 34 long -term development of the soil, which progresses through the release, addition, and 35 36 37 eventual occlusion or loss of minerals and nitrogen. A comprehensive review of nutrient 38 39 cycling is beyond the scope of this paper, but we summarize the most important limits 40 41 - + here. Fixed N (available in the soil as NO 3 and NH 4 ) and minerals P, K, S, Mg, Fe, Mn 42 43 44 are the nutrients required for the production of pigments and enzymes, with N and P 45 46 generally bein g the most limiting nutrients. Nitrogen is the main limiting nutrient, and its 47 48 content in photosynthesizers is the prime correlating variable with photosynthetic 49 50 51 capacity (Schulze, et al. 1994), since the chlorophylls are tetrapyrroles with four nitrogen 52 53 atoms surrounding a magnesium atom (Nobel, 1999). Nitrogen must be fixed originally 54 55 from atmospheric N by enzymatic processes that occur in only particular organisms and 56 2 57 58 59 60 20 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 21 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 form NO - and NH +. Availability is constrained, on land, by ecosystem age (time 4 3 4 5 6 re quired for nitrogen fixing organisms to input nitrogen from the atmosphere into 7 8 developing soil) and losses by leaching or fire. In aquatic and marine environments, 9 10 11 availability is constrained by diffusion from the atmosphere and deposition from land - 12 13 surf ace run -off of organic compounds. Mineral nutrient availability is locally constrained 14 15 by rock substrate, geothermal sources, and deposition from non -local sources. 16 17 18 Phosphorus, an essentialFor mineral Peer for DNA, ATP, Review and phospholipids of cell membranes, 19 20 becom es available from weathering of the mineral apatite but then, over time, complexes 21 22 with Al, Fe, and Mn (at low pH) or with Ca (at high pH), such that it becomes 23 24 25 unavailable. In aquatic or ocean environments, Fe and P in addition to N are the primary 26 27 limiti ng nutrients, and are input via atmospheric deposition or river run -off. For more 28 29 details, the reader is referred to Schlesinger (1997). 30 31 32 33 34 35 36 37 4. Spectral signatures of photosynthetic organisms 38 39 40 41 Given the above information on photosynthesizers’ metabolism, pigm ents, and 42 43 44 environmental niches, how do these features combine into the spectral reflectance of the 45 46 whole organism? Figure 2c shows the surface incident photon fluxes of the Sun/Earth (in 47 48 49 millimoles) with reflectance spectra of an oak leaf, a grass, a moss , and a lichen. On first 50 51 glance, the red edge features appear to show some striking correlations with atmospheric 52 53 absorbance features, particularly oxygen and water bands. We wish to find a 54 55 56 physiological explanation. Physical explanations of land plant spectral signatures are 57 58 59 60 21 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 22 of 69

1 DRAFT , Kiang, Astrobiology 2 3 fairly well understood in some aspects, whereas there is less of such information on other 4 5 6 photosynthesizers. In this section, we first detail the reflectance properties of plant 7 8 leaves. We then compare the reflectance spectra of other photosynthetic organisms to 9 10 11 identify evolutionary pressures that would lead to variations, if any, particularly with 12 13 regard to the “red edge.” 14 15 16 17 18 4.1. ComponentsFor of plant Peer leaf spectral Review reflectance, physical explanation 19 20 21 22 Grant (1987) and Vogelmann (1993) r eview in detail the optical properties of 23 24 25 plant leaves, so only a brief summary from these reviews is provided here. In addition, 26 27 the reader is referred to Tucker (1976, 1978) for discussion of satellite sensor bands for 28 29 monitoring whole vegetation canopi es. 30 31 32 Figure 2c shows the typical reflectance signature of land plants, for which the 33 34 significant features include the green bump in reflectance and the “red edge.” The latter 35 36 37 is so -called because plant photosynthetic pigments absorb strongly in the visib le or PAR, 38 39 which strongly contrasts with high scattering in the NIR due to refraction between leaf 40 41 mesophyll cell walls and air spaces inside the leaf. The wavelength of the “red edge” is 42 43 44 more strictly defined as the inflection point in the slope of the r eflectance between the red 45 46 and NIR, and is sometimes referred to as the “red edge inflection point” or the “red edge 47 48 position.” It falls generally around 700 nm, but the location and steepness may vary 49 50 51 according to the organism’s abundance or thickness (i f sensing over a large area) and 52 53 physiological status; as shown later in this paper, there can be distinct differences 54 55 between organism types. 56 57 58 59 60 22 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 23 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 Using the leaf radiative transfer model of Jacquemoud and Baret (1990), we 4 5 6 illustrate in Figure 3 how a leaf’ s spectral reflectance can vary according to a) leaf 7 8 structure and content of b) water, c) Chl a and b, and c) carbon. The leaf structure is the 9 10 11 main determinant of the high reflectance in the NIR in the overall signature, as leaf 12 13 thickness and interstiti al air spaces between mesophyll cells determine the surfaces off of 14 15 which NIR is scattered. The cell membranes are composed of lipids and proteins, and the 16 17 18 cell walls of celluloseFor and lignin. Peer The refractive Review index in crop plants has been measured 19 20 as 1.333 -1.48, with averages around 1.43 (refractive index of air is 1.0, water 1.333, 21 22 soybean oil 1.48) (Gausman, 1974; wavelength -dependent refractive index: Jacquemoud 23 24 25 and Baret, 1990). Note that, in fact, the cell walls also scatter visible light (Figure 3c, 26 27 gray line with no Chl a & b), but pigments absorb here when present. Water content 28 29 affects reflectance in the longer wavelengths, with strong absorbance bands at ~1400 nm 30 31 32 and ~1900 nm. In fact, the exact locations of these bands in the organism may shift with 33 34 physiological status. Hydration status of a leaf positively affects the NIR reflectance, 35 36 37 since cell turgor affects air spaces within the leaf. The Chl a and b content, of course, 38 39 affects the absorbance in the visible. The carbon density affects jus t the reflectance 40 41 bands in the NIR. The absolute red/NIR contrast, in general, increases for thicker leaves 42 43 44 or multiple layers of leaves or organisms, as more chlorophyll per area will absorb more 45 46 in the visible, and more layers and interstitial air space s increase the surfaces for NIR 47 48 scattering per unit area. A weaker observed contrast with the NIR may be possibly due to 49 50 51 properties of the organism surface, which we will detail later when comparing organisms. 52 53 The model of Jacquemoud and Baret (1990), ba sed on a plate representation of 54 55 the leaf (leaf structure is just a tunable parameter), represents well the vertical variations 56 57 58 59 60 23 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 24 of 69

1 DRAFT , Kiang, Astrobiology 2 3 in the spectral reflectance in response to the given parameters. It does not deal with 4 5 6 horizontal variations (wavelengths of cr itical features) such as: variation in other 7 8 pigments that affect the visible spectrum; observations that the NIR end of the red edge 9 10 11 may broaden in curvature and shift in location; and observations that the water 12 13 absorbance bands may blue - or red -shift w ith physiological status due to, for example, 14 15 water stress or phenological changes (Filella and Penuelas, 1994; Penuelas and Filella, 16 17 18 1998; Karnieli, et al.,For 1999). ThePeer nature of these Review shifts in the NIR reflectance is not well 19 20 studied. 21 22 23 24 25 4.2. Variations in ref lectance spectra among photosynthetic organism types 26 27 28 29 Now, we offer perhaps the first attempt at a comprehensive comparison of 30 31 32 reflectance spectra across photosynthetic taxa to determine whether this will yield further 33 34 insights into how such spectra may h ave evolved. In particular, we focus on how the red 35 36 37 edge varies among organisms, because this is the strongest feature that spectrally 38 39 differentiates photosynthesizers from the background surface or water. The red edge 40 41 begins at about 680 nm, where chlor ophyll a peaks in its absorbance in Photosystem II 42 43 44 (PS II) (antenna of both PS II and PS I absorb almost equally 680 nm light) and then the 45 46 reflectance plateaus around 720 -760 nm. An evolutionary explanation for why these 47 48 features occur at those particula r wavelengths is not settled, particularly with regard to 49 50 51 the NIR reflectance. Our driving questions here are: where does the red edge begin, 52 53 where does it end, and where could it occur on another Earth -like planet, if at all? 54 55 56 57 58 59 60 24 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 25 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 Since the red edge is not merely a step function but has a slope with a bottom in 4 5 6 the visible and a top in the NIR, various workers have tried to quantify the location of the 7 8 red edge to distinguish species or quantify variations due to physiological status. The 9 10 11 first derivative of the reflectance with respect to wavelength is a common measure by 12 13 which to identify the point of maximum slope of the red edge, the “red edge inflection 14 15 point” (REIP). Some workers take this point as the strict definition of the red edge, but 16 17 18 here we w ill use “redFor edge” to Peer encompass the Review span of the rise in reflectance from the 19 20 visible to the NIR. The REIP varies with the level of the red absorbance and NIR 21 22 reflectance, as well as with shifts in the wavelength of the onset of the NIR plateau. 23 24 25 Therefore , we hypothesize that the wavelength of the NIR end may have more physical 26 27 meaning than the red edge inflection point. For this wavelength of onset of the NIR 28 29 plateau, we will coin the term, the “NIR end.” 30 31 32 This NIR end is at least a function of the spectr al spread of pigment absorbance, 33 34 which, as we mentioned earlier, is affected by the proteins in which the pigments are 35 36 37 bound. We are interested in surveying the variation in this NIR end and discerning 38 39 whether it is the result of any environmental adaptat ions. Quantification of the location 40 41 of this NIR end is not entirely straightforward because of the variations in curvature and 42 43 44 slope that can occur over the NIR end and NIR plateau, respectively. We found the third 45 46 derivative of the reflectance with res pect to wavelength to capture fairly well the point at 47 48 which the NIR plateau begins to level off, as this quantifies the “jerk” or change in the 49 50 51 acceleration of the slope. The second derivative identifies better the point just before the 52 53 NIR onset begins, rather than the plateau side. Because these measures are still 54 55 56 57 58 59 60 25 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 26 of 69

1 DRAFT , Kiang, Astrobiology 2 3 somewhat subjective, we also calculate the first derivative as a well -defined measure of at 4 5 6 least relative differences between spectra. 7 8 Presence of the red edge across photosynthetic taxa. Figure 4 shows spectral 9 10 11 reflectance measurements (not a model) of a) land -based vascular plants, b) aquatic 12 13 plants, c) mosses, d) lichens, e) algae, and f) different layers of a microbial mat in an 14 15 alpine lake (species and sources summarized in Appendix A2 ). From this survey, it 16 17 18 appears that all photosynthesizersFor Peer except the purpleReview bacteria (Figure 3d) have a “red 19 20 edge,” albeit this edge is weak in lichens and bacteria. For organisms under water, the 21 22 NIR plateau in the range 760 -850 nm would be absorbed by water and not visible from 23 24 25 above, but apparently, as seen from the aquatic plants’ spectra here, when removed from 26 27 the water, the refractive properties with the air are such that these organisms also have a 28 29 red edge. 30 31 32 In stark contrast, Figure 4e shows that the purple bacteria clearly have no red 33 34 edge, but instead show strong absorbance in the NIR and possibly adjacent NIR “edges.” 35 36 37 The green sulfur exhibit red edge -like reflectance, with variation in the pigments in the 38 39 visible. The orange line is of a mixture of filamentous bacteria, diatoms, and 40 41 precipitations of elemental sulfur; a red -edge feature also appears in this mix. All spectra 42 43 44 in this figure are in situ measurements of whole mat layers (but outside the water) of 45 46 species from the same microbi al mat in an alpine bog pond (Wiggli, et al., 1999; data 47 48 courtesy of Reinhard Bachofen). The purple bacteria are Chromatium species, 49 50 51 anoxygenic photolithoautotrophs with BChl a or BChl b, and engage in sulfide reduction. 52 53 The green sulfur bacteria are al so anoxygenic and utilize BChl a and BChl c, d and e, the 54 55 latter three bacteriochlorophylls having absorption peaks in the range 718 -750 nm. The 56 57 58 59 60 26 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 27 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 colonies may be mixed with oxygenic bacteria, such that the spectrum is not purely green 4 5 6 sulfur bacteria. Mor e measurements are needed to confirm distinct spectra for these 7 8 different kinds of bacteria. Figure 4g shows reflectance spectra of various non - 9 10 11 photosynthetic surfaces, mineral and organic, including human skin and dead grass. 12 13 Hematite and (live) human s kin show some striking similarities to the photosynthetic 14 15 organism spectra in that they have a color in the visible and high reflectance at longer 16 17 18 wavelengths, but which,For are veryPeer different from Review those of the red edge features. 19 20 Horizontal variations in th e NIR end across taxa. Figure 5 shows enlargements 21 22 of the red edge section of the reflectances in Figure 4, with the 761 nm oxygen absorption 23 24 25 line as a reference that reveals differences in the wavelength of the NIR end. As an 26 27 example of how we calculate the NIR end, Figure 6 shows the third derivative of 28 29 reflectance (left plot) and the reflectance spectrum of an aquatic plant. The vertical line 30 31 32 indicates the maximum of the third derivative and the NIR end. Figure 7 shows a scatter 33 34 plot of a) the NIR en d wavelengths of the spectra by organism type, and b) the maximum 35 36 37 slope wavelengths for the same spectra. There are clear trends by organism type: the 38 39 terrestrial plant NIR end is reddest, ranging 746 -765 nm; aquatic plants cluster blue -ward 40 41 of land plan ts at 730 -745 nm; mosses, temperate lichens, and temperate algae range over 42 43 44 720 -733 nm. The snow algae have the bluest red edge. Because of the noisiness of the 45 46 snow algae data, we could not calculate the NIR end, but the maximum slope wavelength 47 48 shows c lear trends. Also, due to interference of atmospheric oxygen, some reflectance 49 50 51 spectra have a spike at 761 nm. 52 53 From this survey, it appears that the NIR reflectance, though common, does not 54 55 appear to be universally the same among all photosynthetic orga nisms, but there appear 56 57 58 59 60 27 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 28 of 69

1 DRAFT , Kiang, Astrobiology 2 3 to be consistent variations among taxa. Lichens often have no sharp edge, but a steady 4 5 6 slope. Purple bacteria have possibly an NIR edge, which is consistent with their pigment 7 8 absorbance spectra. There seems to be a trend in the “NIR end” (where the NIR 9 10 11 reflectance begins to plateau in the red edge), in which the most structurally advanced to 12 13 simplest organisms are ordered from reddest to bluest . More research is needed to 14 15 explain these trends. 16 17 18 For Peer Review 19 20 5. Evolutionary Rationales for Photo synthetic Surface Spectral 21 22 23 Reflectance 24 25 How the properties of pigments, cell membranes, and cell walls evolved is not 26 27 well known, but some evolutionary rationale is needed, with regard to why particular 28 29 30 spectral features of photosynthetic organisms occur a t particular wavelengths, before we 31 32 can conjecture where similar features might arise on another planet. From what we have 33 34 observed of Earth photosynthesizers, it seems especially important to know the 35 36 37 evolutionary pressures on the following features: 1 ) photosynthetic reaction center 38 39 excitation wavelengths, 2) core antenna peak absorbances, 3) accessory pigment peak 40 41 absorbances, 4) beginning and ending wavelengths of the red edge, 5) NIR reflectance 42 43 44 bands. The primary evolutionary pressures or constrai nts may have been chemical or 45 46 thermodynamic, environmental, and ecological. 47 48 49 50 51 52 53 5.1. Pigment absorbance energetics 54 55 56 57 58 59 60 28 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 29 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 Here, we examine why the peaks in pigment absorbance are at their particular 4 5 6 wavelengths, other than due to being in a light transmittance window . 7 8 Minimum energy requirements at the reaction centers: thermodynamics. 9 10 11 Conversion of electronic photon energy to chemical energy occurs at the reaction centers. 12 13 In brief, the energy requirements for this to happen are that the ground state of the 14 15 primar y donor chlorophyll of an RC be at a higher redox potential than the reductant in 16 17 18 order to oxidize it,For and the photon Peer must be of Reviewsufficient energy to excite the primary 19 20 donor to a sufficiently low redox potential so that it can reduce various intermediates to 21 22 reduce the final electron acceptor. So, the RC primary donor must straddle the roles of 23 24 25 both oxidant in its ground state and reductant in its excited state (Blankenship and 26 27 Hartman, 1998). In oxygenic photosynthesis (Equation 2), the excitation, elec tron 28 29 abstraction, and reductions are achieved in a two -step zig -zag series of potential changes 30 31 32 (the “Z -scheme” mentioned earlier), where the reductant H 2O replaces the lost electron 33 34 from the ground state of Chl a (P680 at 680 nm) in PS II, and PS II suppl ies the electron 35 36 37 to replace that in PS I (P700 at 700 nm), which generates the reduced product eventually 38 39 used for fixation of CO 2. P680 of PS II is at a higher potential than the water, allowing it 40 41 to be a strong oxidant of water (Blankenship, 2002; To mmos and Babcock, 2000). 42 43 44 The potential difference of P680 from water results from the molecular configuration of 45 46 the remarkable reaction center of PS II. Note that oxidation of water does not depend on 47 48 the wavelengths of the photons used, but on the mid point redox potential of the oxygen 49 50 51 evolving complex and the reaction center relative to water. In bacterial systems, the 52 53 potential span afforded by RCs absorbing at 800, 850, and up to 960 nm is smaller and 54 55 adequate for these other electron donor/accepto r combinations. But, thus, we are still left 56 57 58 59 60 29 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 30 of 69

1 DRAFT , Kiang, Astrobiology 2 3 with the question, why – on Earth – is P680 at 680 nm, or, more generally, why are the 4 5 6 other reaction centers at their exact wavelengths? If there is no thermodynamic necessity, 7 8 then perhaps the driving force must be environmental pressure on what light can be 9 10 11 harvested. 12 13 Electron transport between light harvesting complexes and reaction center: 14 15 kinetics. In plants and most other photosynthetic organisms, the chlorophylls have peak 16 17 18 absorbances at the Forlongest w avelengthPeer and, hence,Review lowest energy compared to the other 19 20 pigments, which allows the energy cascade via resonance transfer to work. In PS II, the 21 22 reaction center’s (longest) peak absorption wavelength is at 680 nm (P680), and in PS I it 23 24 25 is at 700 nm (P700 ). In anoxygenic bacteria, the known reaction centers are P800 26 27 (heliobacteria), P840 (green sulfur bacteria), P870 (purple bacteria, various sulfur and 28 29 non -sulfur species), P870 (green filamentous, Chloroflexus aurantiacus ), and P960 30 31 32 (Blastochloris virid is , the actual peak is somewhat variable dependent on the solvent or 33 34 core antenna environment) (Ke, 2001; Blankenship and Prince, 1985). The core antennae 35 36 37 are generally integral to the reaction center complex, but may have slightly different 38 39 spectral peak s. The chlorophylls and bacteriochlorophylls also harvest light at a major 40 41 peak in the blue, but the RCs operate at the red peak. There are generally about 300 42 43 44 antenna chlorophylls per RC chlorophyll. 45 46 In green sulfur, green filamentous, heliobacteria, a nd in cyanobacteria, the core 47 48 antennae absorbance spectra peak at shorter wavelengths than the RCs, though there is 49 50 51 considerable spectral overlap. In purple bacteria, the core antennae peak at longer 52 53 wavelengths than the reaction centers (B1015 in B. viri dis whose RC is P960, B890 in 54 55 several purple bacteria that have P870, and B875 in Rhodobacter sphaeroides, whose RC 56 57 58 59 60 30 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 31 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 is P870; Ke, 2001; Scheer, et al., 2003; Richard Cogdell and Andrew Gall, personal 4 5 6 communication, data in Figure 1b; and Blankenship, 2002) . So, interestingly, light 7 8 absorbed at wavelengths longer than the reaction centers can be transferred up the energy 9 10 11 hill to the RCs (Permentier, 2001; Trissl, 1993; Bernhard and Trissl, 2000). The transfer 12 13 mechanism continues to be the subject of debat e and research. In most purple bacteria, 14 15 the spectral overlap of the RCs and core antennae is sufficient such that thermal 16 17 18 variability is enoughFor to prevent Peer exciton energy Review from being trapped in the core antennae; 19 20 however, the wavelength separation between the RC and core antenna peaks of B. viridis 21 22 seems to require some other means of energy transfer. Trissl (1993) proposed a model of 23 24 25 the transfer kinetics and trapping times; assuming a fast thermal equilibration of the 26 27 excitation energy before the charge separation and constraints on quantum yield, the 28 29 model seems to explain that the longer wavelength absorption does not affect the trapping 30 31 32 time or the quantum yield, and it is profitable for the organism since this allows 33 34 utilization of the available ligh t. This seems to provide a sensible explanation for the 35 36 37 uphill exciton transfer from BChl b at 1013 nm to the RC at 980 nm. Mauzerall and co - 38 39 workers (Hou, et al., 2001a; Hou, et al., 2001b; Boichenko, et al., 2001) quantified, in 40 41 detail, the entropy chan ges that occur in PS I, PS II, and cyanobacteria, and found them 42 43 44 to be very small. Work on kinetics of electron transfer (Trissl, 1993; Trissl, et al., 1999; 45 46 Bernhard and Trissl, 2000) implies the optimality of arrangements between the reaction 47 48 centers and light harvesting antennas to ensure efficient transport and trapping of the 49 50 51 excitons. 52 53 The above theoretical work does not allow for prediction of the wavelength of the 54 55 reaction centers, but it does offer an explanation of the excitation energy trans fer kinetics 56 57 58 59 60 31 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 32 of 69

1 DRAFT , Kiang, Astrobiology 2 3 between the reaction centers and light harvesting complexes. The bulk of photosynthetic 4 5 6 activity in B. viridis and other purple bacteria is probably driven by mostly shorter - 7 8 wavelength photons (David Mauzerall, personal communication), but st ill, nature finds 9 10 11 ways to harvest the available light. The light absorbed to 730 nm by oxygenic 12 13 photosynthesizers also gets transferred uphill to the P700 reaction center, but this is just 14 15 the tail end of the absorption spectrum of chlorophyll in P700 and presumed not to 16 17 18 contribute a large amountFor to thePeer total photosynthetic Review activity. However, Krausz, et al. 19 20 (2005) found that there is charge separation in PS II in spinach even over the range 700 - 21 22 730 nm. So, uphill energy transfer is not the most productiv e way to obtain energy, but 23 24 25 nonetheless the phenomenon indicates more means of light harvesting, while resonance 26 27 transfer of energy toward the red is the dominant means of light harvesting and energy 28 29 trapping. 30 31 32 33 34 In general, the literature on light harvesti ng has focused on excitation energy 35 36 37 transfer kinetics, while the literature on reaction centers has focused on figuring out the 38 39 molecular structure and mechanisms, genetic lineage, and molecular evolution (Ferreira, 40 41 et al., 2004; McEvoy, et al., 2005; Xio ng, et al., 2000; Dismukes, et al., 2001). 42 43 44 Meanwhile, theoretical work on the chemical evolution of chlorophyll and the PS II 45 46 oxygen -evolving complex is based on ancient ocean chemistry (Mauzerall, 1976; 47 48 Dismukes, et al., 2001; Blankenship, et al., 1998 ; Dasgupta, et al., 2004) and provides 49 50 51 energetic constraints on the steps toward the development of PS II. Little thus far has 52 53 been done on solar radiation pressures on the evolution of pigment absorbance spectra, so 54 55 we attempt to address such evolutiona ry drivers here. 56 57 58 59 60 32 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 33 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 4 5 6 Atmospheric spectral transmittance. The available light spectrum, of course, 7 8 must be the first constraint on reaction center peak absorbance wavelengths. Numerous 9 10 11 studies on diverse photosynthetic organisms show that the radiation abso rption spectra of 12 13 light harvesting pigments matches the spectrum of incident light in the organism’s 14 15 environmental niche. Examples include: microbial mats at different water strata 16 17 18 (Lengeler, et al., 1999,For Eraso andPeer Kaplan, 2001, Review and Reinhard Bachofen, p ersonal 19 20 communication); purple and green photosynthetic bacteria in NIR and low light 21 22 (Blankenship, et al, 1995); low -light plants (Marschall and Proctor, 2004); red algae and 23 24 25 cyanobacteria absorbing in green wavelengths (Samsonoff and MacColl, 2001); a nd, 26 27 recently discovered, cyanobacteria that perform oxygenic photosynthesis utilizing near - 28 29 infrared radiation (Chen, et al., 2005). 30 31 32 In the wavelength locations of the onset and plateau of the red edge, several 33 34 biological and atmospheric phenomena occu r at both locations. Details of the charts in 35 36 37 Figure 3 are shown in Figure 4 to illustrate more clearly the features around the red edge 38 39 region. The bottom of the red edge varies little from 680 nm, which is the peak 40 41 absorbance wavelength of most of the antenna chorophylls, as well as the primary donor 42 43 44 PSII in plants, algae, and cyanobacteria. . However, depending on measurement 45 46 precision or perhaps cell structures, the onset can be as low as 670 nm (lichen, Licedea -1 47 48 in Fig 4c) and does not appear to go beyond 700 nm, where some of the long -wavelength 49 50 51 forms of antenna chlorophylls absorb. Also, the Chl a primary donor of PS I has its peak 52 53 absorbance at 700 nm. Significant phenomena occur in the red edge region (Krausz, et 54 55 al., 2005, called it “spectral congestion” with regard to an even more detailed structural 56 57 58 59 60 33 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 34 of 69

1 DRAFT , Kiang, Astrobiology 2 3 breakdown than summarized here) at the following wavelengths: 650 -670 nm, where core 4 5 6 antenna minor sub -bands occur for PS I and PS II; 678.5 nm, the main sub -band for PS 7 8 II; 680 nm, the location of the primary donor P680 for PS II; 682 nm, the main sub -band 9 10 11 for PS I; and 700 nm, the primary donor P700 for PS I. The main sub -bands are actually 12 13 where most of the light harvesting of the core antenna occurs. From these data, it appears 14 15 that the max imum absorbance at the foot of the red edge rarely departs from the 678.5 nm 16 17 18 sub -band in organismsFor that utilize Peer PS II. Review 19 20 Perhaps the most significant spectral feature to observe is that the maximum 21 22 photon flux density at the Earth’s surface occurs at 685 n m, just before a drop in 23 24 25 atmospheric transmittance due to oxygen at 687.5 nm. The O 3 Chappuis band (500 -700 26 27 nm) shifts the Sun’s photon spectral flux density from its top -of -the -atmosphere peak at 28 29 600 nm to 685 nm at the Earth’s surface, which may partial ly explain why chlorophyll 30 31 32 favors the red rather than the green. (Note that, a t the Earth’s surface, the incident energy 33 34 flux peak is spread over 450 -490 nm, in the green). Thus, it appears that the peak 35 36 37 absorbance at the foot of the red edge is an adapta tion to harvesting light in the 38 39 atmospheric transmittance window with the most abundant photon flux, and the peak is at 40 41 the most red -shifted limit of that window to afford exciton transfer from accessory 42 43 44 pigments at the shorter wavelengths in that window. The long wavelength limit of this 45 46 window is due to both the solar spectrum and the presence of oxygen, the very product of 47 48 photosynthesis. Note that, if the surface incident light spectrum is viewed in terms of 49 50 51 energy flux rather than photon flux, the pe ak flux is around 480 -490 nm, which is in the 52 53 blue -green; since photosynthesis counts photons, not total energy, it is the peak photon 54 55 flux that is favored by pigments. On the other hand, of course, if photosynthesis evolved 56 57 58 59 60 34 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 35 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 originally under water prior to atmospheric oxygen build -up, then there is no good reason 4 5 6 for the oxygen band to have supplied any evolutionary pressure on the reaction center 7 8 peaks, unless such selective pressure continued to occur on near -surface organisms. 9 10 11 However, this may explai n why PS I and PS II of green cyanobacteria and green algae 12 13 had the advantage in giving rise to land plants. 14 15 The NIR end of the red edge is clearly confined to wavelengths between the 16 17 18 oxygen A -band at For761 nm and Peer the bluest side Reviewof the water band at about 718 nm. Only 19 20 the snow algae have an NIR end blue -shifted from this 718 nm. One might expect 21 22 organisms under ice or water to have spectral characteristics adapted to these media in 23 24 25 contrast to air. However, there is no distinctive feature for ice absor ption or reflectance 26 27 blue -ward of the water band, and for organisms under water, there is not clearly a tight 28 29 relation with the water absorption band in our data. Figure 6b shows our one anoxygenic 30 31 32 example together with the irradiance through water. It m ay be that the peak absorption 33 34 wavelength of this purple bacteria is related to the water band at ~810 -840 nm. More 35 36 37 whole -organism reflectance data are needed to draw any firm conclusions, but 38 39 tentatively, we hypothesize that the NIR end has evolved in re sponse to major absorbance 40 41 bands of the air or water medium of the photosynthesizer. 42 43 44 Purple bacteria have a starkly different reflectance spectrum, with our one 45 46 example showing an “NIR edge” (perhaps better called an “NIR slope”) rather than a red 47 48 edge, s tarting at 837 nm. The bacteriochlorophylls for the purple bacteria and green non - 49 50 51 sulfur bacteria result in primary donor or reaction center wavelengths at 840 to 960 nm. 52 53 Unfortunately, few single -colony or whole -organism reflectance spectra have been 54 55 me asured of anoxygenic bacteria. The data here were measured only over 400 -900 nm, so 56 57 58 59 60 35 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 36 of 69

1 DRAFT , Kiang, Astrobiology 2 3 we cannot examine other NIR features or the spectrum of species like B. viridis that 4 5 6 harvest light at longer wavelengths. 7 8 9 10 11 12 13 5.2. NIR scattering 14 15 16 17 18 As seen in ForFigures 4 - 7,Peer not all photosynthetic Review organisms exhibit the same degree 19 20 of NIR scattering. Why does the NIR reflectance vary, whether due to environmental 21 22 adaptations, physiological status, or other unexplained evidence? 23 24 25 Morphological adaptations to light and climate. It is w ell known that 26 27 morphological adaptation to different climate limits and light levels will influence 28 29 spectral characteristics due to differences in leaf thickness or canopy density. These 30 31 32 quantities affect the boundary layer conductance at the leaf surface and light penetration 33 34 into the leaf (where the leaf boundary layer is the gas diffusive layer of air at the surface). 35 36 37 Thus, cold, dry environments favor needleleaf plants, wet, temperate environments favor 38 39 broadleaf species, and hot, dry environments fav or succulents with low surface -to - 40 41 volume ratios (Holdridge, 1967; Larcher, 1995; Schuepp, 1993; Foley, et al., 1996). 42 43 44 Highly sunlit leaves will be thicker to allow for a greater absorption cross section of 45 46 photosynthetically active radiation (Reich, et al., 1997; Kull, 2002); therefore, the 47 48 visible/NIR contrast will be stronger for high light -adapted leaves. In addition, leaf 49 50 51 surface characteristics, such as hairs or trichomes, specularity of the surface, and surface 52 53 waxes, can affect the overall refle ctance; hairs help increase albedo in hotter, brighter 54 55 environments, while waxes may serve to absorb high UV in arctic environments. 56 57 58 59 60 36 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 37 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 Compactness of cells. A lower NIR reflectance may result from a lack of air 4 5 6 spaces around more compact cells. For examp le, loss of cell turgor during water stress 7 8 could reduce air -cell wall interfaces for NIR scattering. Lichens have a dense structure in 9 10 11 a fungal cortex that overlies their cyanobacterial layer, and therefore, they are not highly 12 13 reflective in the NIR. Co nifer species have a dense mesophyll structure, and hence, their 14 15 leaves tend to be darker in the NIR than those of broadleaf plants. In contrast, Sphagnum 16 17 18 moss increase ratherFor than decrease Peer NIR reflectance Review when dried (dried spectra not shown), 19 20 because the ir means of water supply to the plant’s capitula is not through internal 21 22 conducting cells but through precipitation or capillary rise only (Harris, et al., 2005). 23 24 25 Abundant hyaline cells provide a large water holding capacity in Sphagnum, but their 26 27 drying results in structural changes much different from that of vascular plant leaf 28 29 mesophyll cells. Sphagnum magellicanum (moss Figure 4c) has a very low -sloping red 30 31 32 edge compared to the other mosses, due to more tightly bunched capitula. 33 34 Energy balance. Although the NIR reflectance must play some role in an 35 36 37 organism’s energy balance, it is not clear how important this is in the organism’s survival 38 39 and evolution of its spectral signature. In snow algae, Gorton, et al. (2001) found a 40 41 substantial NIR absorb ance of the outer membrane, which could possibly afford a more 42 43 44 favorable energy balance for the algae. On the other hand, as noted above, the NIR 45 46 reflectance decreases in desiccated plant leaves but increases in dried mosses. Lichens, 47 48 in both cold arctic and hot tropical environments, exhibit very low scattering (reflectance 49 50 51 as well as transmittance) in the NIR (transmittance <15%, Bechtel, et al., 2002). Aquatic 52 53 plants and algae and cyanobacteria that grow under water have no clear need for an NIR 54 55 refle ctance to control their energy balance. The NIR reflectance can be found to vary by 56 57 58 59 60 37 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 38 of 69

1 DRAFT , Kiang, Astrobiology 2 3 the same degree in diverse ecosystems across different environmental gradients, such that 4 5 6 it is not straightforward to draw a conclusion about the NIR reflectance’s role i n an 7 8 organism’s survival or competitive advantage. Undoubtedly, it is important to some 9 10 11 organisms, but there are not yet enough data to determine consistent trends. More studies 12 13 are needed of the relation between the energy balance of photosynthetic orga nisms, their 14 15 climatic limits, and their spectral reflectance signatures. 16 17 18 Cell wall refractiveFor indexPeer and light transmission.Review The cell wall composition of 19 20 organisms, of course, must have a definite functional role in exchange of gas and fluid, 21 22 structural su pport (or not), and transmission of light, and it determines the spectral 23 24 25 refractive index, as mentioned earlier. In Table 1, it can be seen that there are some 26 27 differences between taxa. Terrestrial plant cell walls contain cellulose, lignin, 28 29 polysacchar ides, and protein, whereas aquatic plants contain little or no lignin, since it is 30 31 32 not necessary for support. Algal cell walls are predominantly cellulose; dinoflagellates, 33 34 which are the photosynthetic symbionts in corals and are responsible for algal blo oms 35 36 37 known as red tides, also have a theca, an armor -like set of plates beneath the plasma 38 39 membrane (Larkum and Vesk, 2003; Evitt, 1985). Cyanobacteria cell walls contain 40 41 murein. Unfortunately, the table is incomplete, since there are few data on cell wa ll 42 43 44 compositions for other photosynthetic organisms. The cell wall composition of purple 45 46 bacteria might not have very different refractive properties from that of other 47 48 photosynthesizers, so long as the NIR radiation can be transmitted into the cell. 49 50 51 52 53 6. Con clusions 54 55 56 57 58 59 60 38 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 39 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 To summarize, the full reflectance spectrum of a photosynthetic organism is the 4 5 6 expression of both molecular and macrostructural properties: 1) the absorbance spectra 7 8 of the light -harvesting complex, the core antenna complex, and the reaction c enters, 2) 9 10 11 cell membrane and cell wall refractive properties, and 3) whole -organism structural 12 13 impacts on light scattering. How the above properties evolved is not well known, but a 14 15 number of environmental pressures and molecular constraints play a role: 1) the 16 17 18 thermodynamics ofFor light harvesting Peer and exciton Review transfer kinetics, 2) the redox potential 19 20 requirements for oxidation of the electron donor and reduction of CO 2, 3) adaptation to 21 22 available resources (light spectrum, nutrients, electron donor), which i s not covered in 23 24 25 detail here, 4) adaptation for protection against environmental harm (UV radiation, 26 27 temperature, chemical toxicity, e.g. levels of pH, O 2, other). 28 29 Electron abstraction from the reductant, such as H O, H S, or FeOH +, does not 30 2 2 31 32 depend on the wavelength of the photon but on the redox potential of the biochemical 33 34 molecule. The excitation of the reaction center chlorophyll to a sufficiently energetic 35 36 37 state does depend on the photon energy, and multi -photosystem pathways could 38 39 theoretically util ize more photons at longer wavelengths to evolve O 2 and fix carbon. A 40 41 long wavelength limit might be 1100 nm, a threshold between optical and thermal or 42 43 44 vivartional. The ability to perform electronic transitions, however, depends on the 45 46 molecule, and mor e research is needed to define strict thermodynamic contraints. The 47 48 kinetics of exciton transfer require sufficent proximity between light harvesting pigments 49 50 51 and the reaction center for efficient trapping of the excitons, such that it is more 52 53 energetica lly favorable for the RC to absorb at longer wavelengths; however, uphill 54 55 56 57 58 59 60 39 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 40 of 69

1 DRAFT , Kiang, Astrobiology 2 3 electron transfer does occur. The spectrum of available light ultimately limits pigment 4 5 6 absorption spectra and productivity. 7 8 For the full reflectance spectrum of the organism, w e observed, on Earth, that the 9 10 11 “red edge” is nearly ubiquitous among oxygenic photosynthesizers, but it is weak or 12 13 negligible in lichens. For cyanobacteria, more data are needed. Although it is fairly well 14 15 understood how the NIR reflectance varies due to morphology, the selective role of the 16 17 18 NIR reflectance is Fornot well understood. Peer Thus Review far there is anecdotal evidence with regard 19 20 to the organism’s energy balance (and one study on crop leaves by Aboukhaled, 1966) 21 22 and not enough data on the diversity of cell membrane and cell wall compositions. For 23 24 25 anoxygenic photosynthetic bacteria that have their reaction centers in the NIR, the one 26 27 example for purple bacteria shows no red edge, as it would not make sense for the 28 29 organism to scatter light in the wavelength s that it utilizes. The green sulfur bacteria, 30 31 32 oddly, appear to exhibit a bit of red edge, so more measurements are needed to confirm 33 34 whether this is the general case. The spectra we assembled show striking trends in the 35 36 37 NIR end among organisms, with lic hens, algae, and mosses most blue -shifted, terrestrial 38 39 plants most red -shifted, and aquatic plants in the middle. More data on bacteria and algae 40 41 across environmental gradients, along with consistent measurement of ambient conditions 42 43 44 and assessment of the organisms physiological status, are needed to confirm the strength 45 46 of these trends. 47 48 The Sun’s radiation spectrum and the spectral transmittance of the atmosphere 49 50 51 and water environments are the most important selective pressures on critical points in 52 53 the pigment spectra of photosynthetic organisms. Atmospheric oxygen may have altered 54 55 the atmospheric transmittance spectrum enough to favor PS I and PS II absorbance in the 56 57 58 59 60 40 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 41 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 red. Meanwhile, the example of lichens indicates that not all photosynthesizers will 4 5 6 necessarily have a steeply contrasting reflectance where pigments do not absorb and high 7 8 NIR reflectance is not clearly an energy balance adaptation. So, we cannot conclude yet 9 10 11 how a whole organism’s reflectance spectrum, besides the pigment spectra, is a function 12 13 of environmental adaptation. We do not have enough data on bacteria to draw 14 15 conclusions about their reflectance properties, but the one purple bacteria example 16 17 18 indicates that shiftedFor spectral signaturesPeer are possibleReview for organisms using anoxygeni c 19 20 photosystems. Resonance transfer and exciton transfer kinetics appear to work in concert 21 22 with the available light spectrum to constrain the peak absorbance wavelength of the core 23 24 25 antenna and reaction centers. Given the ability of organisms to transfer l ight energy both 26 27 downhill and uphill to the reaction centers, it may be sufficient just to search for a 28 29 pigment signature within particular atmospheric transmittance windows. 30 31 32 We can, therefore, propose the following candidates for photosynthetic pigment 33 34 peak absorbance wavelengths: 35 36 37 38 39 a. the wavelength of peak incident photon flux within a radiation transmittance 40 41 window, as the main environmental pressure; 42 43 44 45 46 b. the longest wavelength within a radiation window for core antenna or reaction 47 48 center pigments, du e to the resonance transfer of excitation energy and an energy 49 50 51 funneling effect from shorter to longer wavelengths; 52 53 54 55 56 57 58 59 60 41 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 42 of 69

1 DRAFT , Kiang, Astrobiology 2 3 c. the shortest wavelengths within an atmospheric window for accessory 4 5 6 pigments, also due to resonance transfer. 7 8 9 10 11 These hypotheses assume some optimality principle, where the peak photon flux 12 13 wavelength is energetically and ecologically most favorable for survival, and organisms 14 15 will have adapted to this. It may be that accidents, inertia, or the slow stages of evolution 16 17 18 will not yield the optimumFor spectral Peer signature Reviewfor photosynthesizers at the time we 19 20 observe them. For example, on Earth, the inefficiency of Rubisco (the carbon fixing 21 22 enzyme on which all photosynthesis depends and which sometimes is rendered useless 23 24 25 for carbon fixation, be cause it also functions as an oxygenase) is the subject of much 26 27 lament and agricultural biotechnological research (Parry, et al., 2003). Plant reflectance 28 29 of green light is often considered a similarly sub -optimal feature of plants, as there 30 31 32 appears a spe ctral mismatch between solar radiation at the Earth’s surface and the 33 34 absorption peaks (440 and 680 nm) of chlorophyll (Raven and Wolstencroft, 2002). 35 36 37 Even with our rules above for pigment properties, there appears to be wasted green light. 38 39 However, give n that algae and cyanobacteria utilize phycobilins to harvest green light, 40 41 the low harvesting of green light in land plants has been thought by some to be an 42 43 44 inefficiency due to an evolutionary “lock -in” from the lineage of green algae. However, 45 46 above -gro und plants pigments do absorb green light, just in a lesser ratio to other colors, 47 48 and they often experience too much light (or are limited by other resources), hence the 49 50 51 need for quenching by carotenoids. The non -light harvesting pigment anthocyanin, 52 53 whi ch accumulates at the surface of shade -adapted leaves and makes them red, may 54 55 provide photo -protection under high light by shading Chl b in chloroplasts from green 56 57 58 59 60 42 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 43 of 69 Astrobiology

1 DRAFT , Kiang, Astrobiology 2 3 light (Gould, et al., 2002; Pietrini, et al., 2002). So, there may be no selective advanta ge 4 5 6 to absorbing more green light. That all land plants are descended from the green algae 7 8 may very much be because their pigment combinations provided the selective advantage 9 10 11 for life on land after the build -up of atmospheric O 2, which shifted the surface spectral 12 13 photon flux from a peak at 600 nm to 685 nm. So, given some caveats about 14 15 evolutionary optimality with regard to light resource constraints, we propose the above 16 17 18 rules for wavelengthsFor of peak photosyntheticPeer Reviewpigment radiation absorbance, dependen t on 19 20 the spectral photon flux density at the surface of a planet. 21 22 To explain whole -organism spectral reflectance and the steepness of absorbance 23 24 25 peaks, more studies are needed of: bacteria spectral properties; the role of pigment - 26 27 protein complexes in alt ering pigment absorbance spectra; thermodynamic limits of light 28 29 harvesting and redox biochemistry; field studies and modeling of organism radiative 30 31 32 transfer, growth, and energy balances within their respective light environments; and 33 34 further speculation on the environment of the early Earth. 35 36 37 38 39 Acknowledgments 40 41 42 Numerous people kindly contributed data and valuable advice for this paper. We 43 44 are greatly obliged to Niels -Ulrik Frigaard, Richard Cogdell, and Andrew Gall for 45 46 47 pigment data and valuable comments; Mi chael Eastwood, Robert Green, and Scott Nolte 48 49 of the JPL/AVIRIS lab for the use of their spectroradiometer; Mike Schaadt and the 50 51 52 Cabrillo Marine Aquarium for providing marine algae samples. Holly Gorton for snow 53 54 algae data; Suzanne Fyfe for seagrass spe ctra; Robert Bryant and Angela Harris for 55 56 moss spectra; Greg Asner for lichen spectra from Hawaii; Reinhard Bachofen for 57 58 59 60 43 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 44 of 69

1 DRAFT , Kiang, Astrobiology 2 3 microbial mat bacteria data; Brian Cairns, Judith Lean, and Andrew Lacis for solar 4 5 6 spectral photon flux densities; Dennis Clark for Hawaii buoy radiation data; Stephane 7 8 Jacquemoud for the use of the PROSPECT model; Warwick Hillier, Yongqin Jiao, and 9 10 11 Elmars Krausz for very helpful explanations about photochemistry. Thanks are also due 12 13 to John Scalo, Norm Sleep, and Jim Kasting for ma ny lively discussions and helpful 14 15 references. We also are grateful to the many people who have made their datasets 16 17 18 available on -line andFor who are citedPeer in this paper. Review Finally, we greatly appreciate the very 19 20 helpful comments of two anonymous reviewers. M. C. thanks NASA for supporting his 21 22 participation in this work through JPL contract 1234394 with UC Berkeley. Govindjee 23 24 25 thanks the Dept. of Plant Biology of the University of Illinois for office support. N.Y.K 26 27 also thanks NASA and James Hansen for supporti ng this work. 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 44 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 45 of 69 Astrobiology

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1 DRAFT, Kiang, Astrobiology 2 3 Macmillan. 4 5 Wiggli, M., A. Smallcombe and R. Bachofen (1999). "Reflectance spectroscopy and laser 6 confocal microscopy as tools in an ecophysiological study of microbial mats in an alpine 7 bog pond." Journal of Microbiological Methods 36(3): 173-182. 8 Wolstencroft, R. D. and J. A. Raven (2002). "Photosynthesis: Likelihood of Occurrence and 9 Possibility of Detection on Earth-like Planets." Icarus 157: 535-548. 10 11 Woodward, F. I. (1987). Climate and Plant Distribution. New York, Cambridge University 12 Press. 13 Wydrzynski, T. J. and K. Satoh (2005). Photosystem II: water: Plastoquinone 14 Oxidoreductase. Dordrecht, Springer. 15 Xiong, J., W. M. Fischer, K. I. Inoue, M. Nakahara and C. E. Bauer (2000). "Molecular 16 evidence for the early evolution of photosynthesis." Science 289: 1724-1730. 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 53 of 69 Astrobiology

1 2 3 4 5 6 7 Table captions 8 9 10 11 12 Table 1. Range of phototrophic organisms on Earth. Bacteria compiled by Janet 13 14 Siefert. Additional carbon sources and electron donors from Overmann and 15 16 17 Garcia-Pichel (2000), Decker, et.al. (2005), Blankenship, et al. (1995), Eraso and 18 For Peer Review 19 Kaplan (2001), Miller, et al. (2005). Algae from Larkum, et.al, (2003). Plants 20 21 from (Nobel, 1999). Abundance and productivity numbers from Reeburgh (1997) 22 23 24 and Cramer, et al. (1999). Bacterial abundance and productivity are autotrophic 25 26 only, primarily marine cyanobacteria, estimated from Whitman, et al. (1998). 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 54 of 69

1 2 3 4 5

6 Table 1. Range of phototrophic organisms on Earth. Bacteria compiled by Janet Siefert. Additional carbon sources and electron donors from Overman and Garcia-Pichel (2000), Decker, et.al. (2005), Blankenship, et al. (1995), Eraso and Kaplan (2001), 7 Miller, et al. (2005). Algae from Larkum, et.al, (2003). Plants from (Nobel, 1999). Abundance and productivity numbers from Reeburgh (1997) and Cramer, et al. (1999). Bacterial abundance and productivity are autotrophic only, estimated from Whitman, et al. (1998). 8 Global Gross 9 Appeared Taxon Growth mode/form Cell wall RC Pigments e-donor C source* Products Niche Abundance Productivity (Pg-C) (Pg-C/yr) 10 BACTERIA unicellular 0.06 51

11 3.8 Ga Anoxygenic 12 dense microbial mats in hot springs often in Green non-sulfur anoxygenic photoorganoheterotroph BChl a/c/ and association with cyano, thermophilic; floculent Type II sulfide orgC; CO2 S 13 filamentous aerobic chemoorganoheterotroph or d/e +car surface layer in alkaline springs; Chloroflexus: For Peer Reviewmax ~70 Celsius, cannot fix N, resistant to UV 14 orgC: acetate, BChl a + sulfide, reduced S, non-thermal aquatic ecosystems, hot springs, Green sulfur bacteria anoxygenic photolithoautotroph Type I propionate, sulfate 15 c/d/e +car H2, Fe max 55-56 Celsius (Chlorobium tepidum) pyruvate; CO2 inorg&orgC, S, S, fresh and marine waters, eutrophic marine, hot 16 aerobic and anoxgenic heterotrophs BChl a/b 3.8 Ga Purple bacteria Type II sulfate, sulfide, orgC, CO sulfate, springs, anoxic aquatic sediments; max >50 17 and anoxygenic autotroph +car 2 sulfite, H2, Fe CO2 Celsius (Chromatium tepidum) pyruvate, ethanol, soil, dry paddy fields, occasionally lakeshore 18 sulfide, reduced S, Heliobacteria anoxygenic photoorganoheterotroph Type I BChl g+car lactate, acetate, and ? muds, hot springs; resistant to UV, fix N; sulfate 19 butyrate survive at least to 42 Celsius Halobacteria C5 isoprenoid chains bacterio- bacterio- salt crusts in marine salterns, saline lakes, 20 (not actual aerobic chemoroganoheterotroph attached to glycerol N/A orgC ? rhodopsin rhodopsin evaporites, ~4M NaCl 21 photosynthesis) by ether linkages 3.6-2.3 Ga Oxygenic 22 Chl a/b/c/d 3.6-2.3 Ga Cyanobacteria oxygenic photolithoautotroph murein Type I & II H O, S CO O everywhere, -15 to +75ºC ? ? 23 +PBS+car 2 2 2 eukaryotes, autotrophs, unicellular, 1.2 Ga ALGAE Type I & II H O, other? CO O fresh and marine waters, snow 2 ~100 24 multicelluar 2 2 2 Rhodophytes Chl a + PBS min observed PAR flux 0.01 micromol/m2/s 1.2 Ga cellulose, xylan 25 (red algae) + car (Littler, et.al., 1986) 26 Chromophytes cellulose; diatoms: " Chl a/c + car 27 (incl. brown algae) silica, alginate Chlorophytes Chl a/b/c 750 Ma cellulose 28 (green algae) +PBS +car Lichens 29 symbiosis of fungus and crustose, squamose, Chl a/b+car H O rock outcrops, vegetation surfaces ? ? cyanobacteria/algae 2 30 foliose, fruticose 31 cellulose, lignin, polysaccharides (e.g. O2, min observed PAR PLANTS leaves, stems, roots Type I & II Chl a/b+car H2O CO2 550-680 ~90-120 32 pectin), protein, VOCs ~ 3 micromol/m2/s (0.7 W/m2) water, calcium 33 460 Ma Bryophytes non-vascular moist land environments 34 Mosses Sphagnum, Acrocarpus, Pleurocarpus Liverworts 35 Vascular plants broadleaf, needleaf, herbaceous, aquatic to desert environments succulent Aquatic plants - 36 no lignin CO2, HCO3 C3 pathway 37 144 Ma Flowering plants 38 70-55 Ma CAM 20-35 Ma C4 pathway 39 Ga - billion years ago, Ma - million years ago, RC-reaction center, PBS-phycobilisomes, car-carotenoids, VOC-volatile organic compound. *C source related only to the photosynthetic process (disregards carnivorous plants). 40 41 42 43 44 45 46 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 55 of 69 Astrobiology

1 2 3 4 5 6 7 Figure Captions 8 9 10 Figure 1. Electron transport pathways of photosynthesis, with midpoint redox 11 12 potentials of ground and excited states of the reaction centers, of biochemical 13 14 intermediates and reduced products. Shown are photosystems for purple bacteria, 15 16 17 green sulfur bacteria, and oxygenic photosynthesis. 18 For Peer Review 19 20 21 Figure 2. 22 23 24 a) Solar spectral photon flux densities at the top of the Earth's atmosphere and at 25 26 the Earth's surface, and estimated in vivo absorption spectra of photosynthetic 27 28 29 pigments of plants and algae. Sources : Modeled photon flux densities from the 30 31 following: Top-of-the-atmosphere (TOA) irradiance: 150-200 nm, Andrew Lacis, 32 33 NASA Goddard Institute for Space Studies (GISS); 200-400 nm, Judith Lean 34 35 36 (Naval Research Laboratory); 400-2500 nm, Brian Cai rns, NASA GISS. Surface 37 38 irradiance: 200-400 nm, J. Lean (Lean and Rind, 1998); 400-2500 nm, Brian 39 40 Cairns. Hawaii buoy measurements from Dennis Clark (NOAA). Chlorophyll a 41 42 43 and Chlorophyll b absorbance measurements, made by Junzhong Li (H. Du and 44 45 cowork ers, 1998), in vitro , were shifted in wavelengths to match in vivo peaks, 46 47 48 and absorbances were normalized to between 0 and 1. Carotenoid absorption 49 50 spectra are estimated in vivo absorption spectra in green algae (Govindjee, 1960). 51 52 Phycoerythrin and phycoc yanin absorption spectra are unpublished absorption 53 54 55 spectra from Govindjee’s laboratory, and from Ke (2001). Chlorophyll a 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 56 of 69

1 2 3 4 5 6 7 fluorescence spectrum, from spinach chloroplasts, is from Govindjee and Yang 8 9 (1966). Pigments, measurement method, and sources are listed in Appendix A1. 10 11 12 13 14 b) Solar spectral photon flux densities at the top of the Earth's atmosphere, at the 15 16 Earth's surface, at 5 cm depth in pure water, and at 10 cm depth of water with an 17 18 arbitrary concentrationFor of brownPeer algae; algaeReview and bacteria pigme nt absorbance 19 20 21 spectra. Sources: Top-of -the-atmosphere (TOA) and surface incident radation 22 23 same as Figure 2a. Water spectral absorption coefficient: 200-380 nm, Segelstein 24 25 (1981); 380-640 nm, Sogandares, et al. (1997); 640 -2500 nm, Kou, et al., (1993). 26 27 28 Algae (brown, kelp, Macrocystis pyrifera ) absorption coefficient from reflectance 29 30 spectrum measured (in lab, in air) by N.Y. Kiang with ASD FieldSpec 31 32 33 spectroradiometer (instrument from JPL/AVIRIS Lab). Bacteriochlorophyll 34 35 pigment absorbance spectra are all in vivo in intact membranes, including 36 37 carotenoids. BChl a (Rhodobacter sphaeroides ) and BChl b (Blastochloris 38 39 40 viridis ) spectra from Richard Cogdell and Andrew Gall. BChl c, d, and e spectra 41 42 from green sulfur bacteria (Frigaard, et al., 2004). Pigments, measurement 43 44 method, and sources are listed in Appendix A1. 45 46 47 48 49 c) Solar spectral photon flux densities at the top of the Earth's atmosphere (TOA) 50 51 and at the Earth's surface, with reflectance spectra of terrestrial plants, moss, and 52 53 54 lichen (source: Clark, et al., 2003), and O2 and H2O absorbance lines. 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 57 of 69 Astrobiology

1 2 3 4 5 6 7 8 9 10 Figure 3. Modeled reflectance spectra of a generalized plant leaf, from the model 11 12 PROSPECT (Jacquemoud and Baret, 1990). Variations in reflectance due to: a) 13 14 15 structure, b) water content, c) Chl a and b co ntent, and d) carbon content. 16 17 18 For Peer Review 19 20 Figure 4. Reflectance spectra of different photosynthetic organisms, minerals, 21 22 and non-photosynthetic organic matter in 0.2-2.4 micron range. The vertical 23 24 dotted line in all the plots is at 0.761 micron, corresponding to an oxygen 25 26 27 absorption line. Sources : a) Land plants: Clark, et al. (2003). Surfgrass, aquatic 28 29 plant: N.Y. Kiang. . b) Aquatic plants: Fyfe, et al. (2003); surfgrass: N.Y. 30 31 Kiang. c) Mosses: Lang, et al. (2002), Harris, et al., (2005). d) Lichens: Cla dina 32 33 34 and Sterocaulon, courtesy of Greg Asner; Acarospora, Licedea, Xanthoparmelia, 35 36 and Xanthoria, Clark, et al. (2003). e) Algae: N. Kiang, snow algae: Gorton, et 37 38 al. (2001). f) Bacteria in a microbial mat: from Reinhard Bachofen in Wiggli, et 39 40 41 al. (1999). g) Minerals and golden dry grass: Clark, et al. (2003). Human skin: 42 43 N.Y. Kiang. Species names and instruments used are listed in Appendix A2. 44 45 46 47 48 Figure 5. Details of Figure 4. Reflectance spectra of different photosynthetic 49 50 organisms, minerals, and non-photosynthetic organic matter over 0.4-0.9 microns. 51 52 53 Sources : Same as for Figure 4. a) Land plants: Clark, et al. (2003). Surfgrass, 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 58 of 69

1 2 3 4 5 6 7 aquatic plant: N.Y. Kiang. b) Aquatic plants: Fyfe, et al. (2003); surfgrass: N. 8 9 Y. Kiang. c) Mosses: Lang, et al. (2002) ), Harris, et al., (2005). d) Lichens: 10 11 Clark, et al. (2003). e) Algae: N.Y. Kiang, snow algae: Gorton, et al. (2001). f) 12 13 14 Bacteria in a microbial mat: from Reinhard Bachofen in Wiggli, et al. (1999). 15 16 17 18 Figure 6. CalculationFor of thePeer NIR end wa velength,Review aquatic plant, Posidiana 19 20 rd 21 australis . Vertical solid thin line is location of 3 derivative maximum and NIR 22 23 end of the spectral reflectance (data courtesy of Susan Fyfe). 24 25 26 27 28 Figure 7. Scatterplot of wavelengths of the red edge inflection point (“ed ge 29 30 wavelengths”) and the NIR end (“plateau wavelengths”) for organisms in Figures 31 32 33 4 and 5. Vertical axis has no scale, but points are dithered vertically simply to 34 35 show their horizontal spread. 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 59 of 69 Astrobiology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Figure 1- Electron transport pathways of photosynthesis, with midpoint redox potentials 34 of ground and excited states of the reaction centers, of biochemical intermediates and 35 reduced products. Shown are photosystems for purple bacteria, green sulfur bacteria, and oxygenic photosynthesis. 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 60 of 69

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Figure 2- a) Solar spectral photon flux densities at the top of the Earth's atmosphere and 49 at the Earth's surface, and estimated in vivo absorption spectra of photosynthetic 50 pigments of plants and algae. Sources: Modeled photon flux densities from the following: Top-of-the-atmosphere (TOA) irradiance: 150-200 nm, Andrew Lacis, NASA Goddard 51 Institute for Space Studies (GISS); 200-400 nm, Judith Lean (Naval Research 52 Laboratory); 400-2500 nm, Brian Cairns, NASA GISS. Surface irradiance: 200-400 nm, J. 53 Lean (Lean and Rind, 1998); 400-2500 nm, Brian Cairns. Hawaii buoy measurements 54 from Dennis Clark (NOAA). Chlorophyll a and Chlorophyll b absorbance measurements, 55 made by Junzhong Li (H. Du and coworkers, 1998), in vitro, were shifted in wavelengths 56 to match in vivo peaks, and absorbances were normalized to between 0 and 1. Carotenoid 57 absorption spectra are estimated in vivo absorption spectra in green algae (Govindjee, 1960). Phycoerythrin and phycocyanin absorption spectra are unpublished absorption 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 61 of 69 Astrobiology

1 2 3 spectra from Govindjee's laboratory, and from Ke (2001). Chlorophyll a fluorescence 4 spectrum, from spinach chloroplasts, is from Govindjee and Yang (1966). Pigments, 5 measurement method, and sources are listed in Appendix A1. b) Solar spectral photon 6 flux densities at the top of the Earth's atmosphere, at the Earth's surface, at 5 cm depth 7 in pure water, and at 10 cm depth of water with an arbitrary concentration of brown 8 algae; algae and bacteria pigment absorbance spectra. Sources: Top-of-the-atmosphere 9 (TOA) and surface incident radation same as Figure 2a. Water spectral absorption coefficient: 200-380 nm, Segelstein (1981); 380-640 nm, Sogandares, et al. (1997); 640- 10 2500 nm, Kou, et al., (1993). Algae (brown, kelp, Macrocystis pyrifera) absorption 11 coefficient from reflectance spectrum measured (in lab, in air) by N.Y. Kiang with ASD 12 FieldSpec spectroradiometer (instrument from JPL/AVIRIS Lab). Bacteriochlorophyll 13 pigment absorbance spectra are all in vivo in intact membranes, including carotenoids. 14 BChl a (Rhodobacter sphaeroides) and BChl b (Blastochloris viridis) spectra from Richard 15 Cogdell and Andrew Gall. BChl c, d, and e spectra from green sulfur bacteria (Frigaard, et 16 al., 2004). Pigments, measurement method, and sources are listed in Appendix A1. c) 17 Solar spectral photon flux densities at the top of the Earth's atmosphere (TOA) and at the Earth's surface, with reflectance spectra of terrestrial plants, moss, and lichen (source: 18 Clark,For et al., Peer2003), and O 2 andReview H 2O absorbance lines. 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 62 of 69

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Figure 3- Modeled reflectance spectra of a generalized plant leaf, from the model 49 PROSPECT (Jacquemoud and Baret, 1990). Variations in reflectance due to: a) structure, 50 b) water content, c) Chl a and b content, and d) carbon content. 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 63 of 69 Astrobiology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Figure 4- Reflectance spectra of different photosynthetic organisms, minerals, and non- 49 photosynthetic organic matter in 0.2-2.4 micron range. The vertical dotted line in all the 50 plots is at 0.761 micron, corresponding to an oxygen absorption line. Sources: a) Land plants: Clark, et al. (2003). Surfgrass, aquatic plant: N.Y. Kiang. . b) Aquatic plants: Fyfe, 51 et al. (2003); surfgrass: N.Y. Kiang. c) Mosses: Lang, et al. (2002), Harris, et al., (2005). 52 d) Lichens: Cladina and Sterocaulon, courtesy of Greg Asner; Acarospora, Licedea, 53 Xanthoparmelia, and Xanthoria, Clark, et al. (2003). e) Algae: N. Kiang, snow algae: 54 Gorton, et al. (2001). f) Bacteria in a microbial mat: from Reinhard Bachofen in Wiggli, et 55 al. (1999). g) Minerals and golden dry grass: Clark, et al. (2003). Human skin: N.Y. Kiang. 56 Species names and instruments used are listed in Appendix A2. 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 64 of 69

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Figure 5- Details of Figure 4. Reflectance spectra of different photosynthetic organisms, 49 minerals, and non-photosynthetic organic matter over 0.4-0.9 microns. Sources: Same as 50 for Figure 4. a) Land plants: Clark, et al. (2003). Surfgrass, aquatic plant: N.Y. Kiang. b) Aquatic plants: Fyfe, et al. (2003); surfgrass: N. Y. Kiang. c) Mosses: Lang, et al. (2002) ), 51 Harris, et al., (2005). d) Lichens: Clark, et al. (2003). e) Algae: N.Y. Kiang, snow algae: 52 Gorton, et al. (2001). f) Bacteria in a microbial mat: from Reinhard Bachofen in Wiggli, et 53 al. (1999). 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 65 of 69 Astrobiology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Figure 6- Calculation of the NIR end wavelength, aquatic plant, Posidiana australis. 49 Vertical solid thin line is location of 3rd derivative maximum and NIR end of the spectral 50 reflectance (data courtesy of Susan Fyfe). 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 66 of 69

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Figure 7- Scatterplot of wavelengths of the red edge inflection point (
edge 49 wavelengths
) and the NIR end (
plateau wavelengths
) for organisms in Figures 4 50 and 5. Vertical axis has no scale, but points are dithered vertically simply to show their horizontal spread. 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 67 of 69 Astrobiology

1 2 3 4 5 6 7 Appendix A1. Photosynthetic pigment absorbance spectra 8 Pigment Object measured Source 9 Chl a in vitro spectra were In vitro spectra from 10 stretched/shifted in Junzhong Li (H. Du 11 wavelengths to match in and coworkers, 1998) 12 vivo peaks by linear 13 transformation 14 Chl b in vitro spectra were In vitro spectra from 15 stretched/shifted in Junzhong Li (H. Du 16 wavelengths to match in and coworkers, 1998) 17 vivo peaks by linear transformation 18 For Peer Review 19 BChl a intact membranes in Richard Cogdell and Rhodobacter sphaeroides Andrew Gall 20 (pers. comm.) 21 22 BChl b intact membranes in Richard Cogdell and 23 Blastochloris viridis Andrew Gall (pers. comm..) 24 25 BChl c green sulfur bacteria Frigaard, et.al., 26 (2004) 27 BChl d green sulfur bacteria Frigaard, et.al., 28 (2004) 29 BChl e green sulfur bacteria Frigaard, et.al., 30 (2004) 31 phycoerythrin unpublished absorption Govindjee 32 spectra from Govindjee’s (unpublished), and 33 laboratory (Beckman DU Ke (2001) 34 spectrophotometer) 35 36 phycocyanin unpublished absorption Govindjee 37 spectra from Govindjee’s (unpublished), and laboratory (Beckman DU Ke (2001) 38 spectrophotometer) 39 40 carotenoid estimated in vivo Govindjee (1960) 41 absorption spectra in green algae. NOTE: 42 Type of carotenoid not 43 specified. 44 Chl a spinach chloroplasts Govindjee and Yang, 45 flurorescence - (1966) 46

47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 68 of 69

1 2 3 4 5 6 Appendix A2. 7 Photosynthetic organisms reflectance data 8 Organism type Species measured Instrument Resolution Source 9 Terrestrial Engelmann spruce ASD 3 nm (350 -1000 Clark, et al. (2003) 10 vascular plants, lawn grass FieldSpec nm USGS splib05a 11 temperate lodgepole pine 10 nm (1000 - spectral database 12 maple 2500 nm) 13 oak 14 piñon pine walnut 15 16 Aquatic Phyllospadix torreyi ASD 3 nm:.35 -1 µm Nancy Kiang 17 vascular plants FieldSpec 350 - 10 nm:1 -2.5 µm – Calif. coast 2500P 18 ForZostera capricorn Peeri Review 19 Aquatic ASD 3 nm:.35 -1 µm Fyfe, et al. (2003) vascular plants, Posidonia australis FieldSpec FR 20 10 nm:1 -2.5 µm seagrass Halophila ovalis 21 22 Lichens – Acarospora ASD 3 nm:.35 -1 µm Clark, et al. (2003) Licedea 23 temperate FieldSpec 10 nm:1 -2.5 µm USGS splib05a Xanthoparmelia spectral database 24 Xanthoria 25 Cladina skottsbergii 26 Lichens – ASD 3 nm:.35 -1 µm Courtesy of Greg tropical Stereocaulon rocellodies FieldSpec Asner 27 10 nm:1 -2.5 µm 28 Moss – Dicranum ASD 3 nm:.35 -1 µm Clark, et al. (2003 ) 29 temperate Plagiochila FieldSpec 10 nm:1 -2.5 µm USGS splib05a 30 Polytrichum spectral database 31 Moss – Sphagnum capifollium ASD 1 nm: Harris, et al. (2005) 32 Temperate Sphagnum cuspidatum FieldSpec Pro .35 -2.5 µm 33 Sphagnum pulchrum 34 Sphagnum magellanicum 35 Sphagnum papillosum 36 Algae – Calif. Brown -Macrocystis ASD 3 nm:.35 -1 µm Nancy Kiang 37 coast pyri fera FieldSpec 350 - 10 nm:1 -2.5 µm 38 Red – unknown 2500P 39 Green – Ulva labata 40 Algae – snow Chlamydomonas nivalis Ocean Optics 0.66 nm @ Gorton, et al. (2001) 41 (Bauer) Wille S-2000, #754 28 -.86 µm 42 Bacteria Green filamentous ASD LabSpec 1.5 nm Reinhard Bachofen 43 Purpl e sulfur VNIR -512 w/ (pers. comm.) 44 optical fiber Wiggli, et al. (1999) 45 (LDG -GC 46 600/ 750, 25 47 m, Fujikuro, 48 Tokyo, Japan) 49 Bacteria Rhodobacter sphaeroides 0.5 nm Richard Cogdell, 50 Blastochloris viridis Andrew Gall 51 (pers . comm.) 52 Abbreviations: 53 ASD: Analytical Spectral Devices, Inc. 54 USGS: United State Geological Survey 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 69 of 69 Astrobiology

1 2 3 4 5 6 7 8 Appendix A3. Spectral transmittance of light through algae in water in 9 10 Figure 2b 11 12 In Figure 2b, the light incident at a depth of 5 cm in water with algae was 13 14 15 calculated by estimating an absorbance coefficient from a measured reflectance 16 17 spectrum, scaling this absorbance coefficient to approximate a density of algae, 18 For Peer Review 19 20 and then calculating the light transmitted through, given the surface incident 21 22 radiation in Figure 2b. The attenuated light, I(z, λ), at a depth of z cm at 23 24 wavelength λ, given incident light at the surface of I(0, λ), spectral absorption 25 26 27 coefficient of water αwater (λ) and of algae αalgae (λ) with a density parameter ρalgae , 28 29 is: 30 31 32 33

34 −(αwater +ρ a lg ae αa lg ae )z 35 Iz(),λ = I()0, λ e ( 6 ) 36 37 38 39 40 For αwater (λ), we used water spectral absorption coefficient values as 41 42 measured by the following sources: 200 -380 nm, Segelstein (1981); 380 -640 nm, 43 44 45 Sogandares, et al. (1997); 640 -2500 nm, Kou, et al., (1993). 46 47 For αalgae (λ), we took spectral reflectance measurements of fresh samples 48 49 of a brown algae, Macrocystis pyrifera (kelp) w ith a FieldSpec Pro 50 51 52 spectroradiometer (borrowed from Mike Eastwood and company at the JPL 53 54 AVIRIS lab). Since we could not measure transmittance directly, we estimated 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Astrobiology Page 70 of 69

1 2 3 4 5 6 7 transmittance from comparison to the behavior of a clover leaf modeled by the 8 9 PROSPECT l eaf radiative transfer model of Jacquemoud and Baret (1990). A 10 11 leaf transmittance spectrum is almost exactly the same as the reflectance, with the 12 13 14 sum of reflectance and transmittance scattering nearly all NIR near the red edge. 15 16 Therefore, we estimated t he kelp transmittance from a scaling of the reflectance to 17 18 allow for aboutFor 5% absorbed Peer NIR at the redReview edge, and then calculated the spectral 19 20 21 absorbance as 1 - (reflectance + transmittance). To approximate the density of the 22 23 algae in water, we simply let ρalgae = 0.10. 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801