Circular spectropolarimetric sensing of higher plant and algal chloroplast structural variations
C.H. Lucas Patty, Freek Ariese, Wybren Jan Buma, Inge Loes ten Kate, Rob J.M. van Spanning, Frans Snik
CCircular spectropolarimetric sensing of higher plant andalgal chloroplast structural variations
C.H. Lucas Patty , Freek Ariese , Wybren Jan Buma , Inge Loes ten Kate ,Rob J.M. van Spanning , Frans Snik *[email protected] Abstract
Photosynthetic eukaryotes show a remarkable variability in photosynthesis, in-cluding large differences in light harvesting proteins and pigment composition.
Invivo circular spectropolarimetry enables us to probe the molecular architectureof photosynthesis in a non-invasive and non-destructive way and, as such, canoffer a wealth of physiological and structural information. In the present studywe have measured the circular polarizance of several multicellular green, red andbrown algae and higher plants, which show large variations in circular spectropo-larimetric signals with differences in both spectral shape and magnitude. Manyof the algae display spectral characteristics not previously reported, indicatinga larger variation in molecular organization than previously assumed. As thestrengths of these signals vary by three orders of magnitude, these results alsohave important implications in terms of detectability for the use of circularpolarization as a signature of life.
Keywords
Circular polarization, photosynthesis, chloroplast, chlorophyll, algae 1/25 a r X i v : . [ q - b i o . B M ] A ug Introduction
Terrestrial biochemistry is based upon chiral molecules. In their most simpleform these molecules can occur in a left-handed and a right-handed versioncalled enantiomers. Unlike abiotic systems, nature almost exclusively uses thesemolecules in only one configuration. Amino acids, for instance, primarily occurin the left-handed configuration while most sugars occur in the right-handedconfiguration. This exclusive use of one set of chiral molecules over the other,called homochirality, therefore serves as a unique and unambiguous biosignature[1]. Many larger, more complex biomolecules and biomolecular architectures arechiral too and the structure and functioning of biological systems is largelydetermined by their chiral constituents. Homochirality is required for processesranging from self-replication to enzymatic functioning and is therefore also deeplyinterwoven with the origins of life.The phenomenon of chirality, i.e. the molecular dissymmetry of chiralmolecules, causes a specific response to light [2, 3]. This response is bothdependent on the intrinsic chirality of the molecular building blocks and on thechirality of the supramolecular architecture. Polarization spectroscopy enablesthese molecular properties to be probed non-invasively from afar and is thereforeof great value for astrobiology and the search for life outside our solar system.Polarization spectroscopy also has a long history in biological and chemicalsciences. Circular dichroism (CD) spectroscopy utilizes the differential electronicabsorption response of chiral molecules to left- and, right-handed circularlypolarized incident light and is very informative for structural and conformationalmolecular dynamics. As such it has proven to be an indispensable tool in(bio-)molecular research.Chirality can also be observed in chlorophylls and bacteriochlorophylls utilizedin photosynthesis. While their intrinsic CD signal is very weak due to theiralmost planar symmetrical structure, these chlorophylls are organized in achiral supramolecular structure that greatly enhances these signals [4]. This isparticularly the case for the photosynthetic machinery in certain eukaryotes,where photosynthesis is carried out in specialized organelles, chloroplasts, whichin higher plants have a large molecular density yielding anomalously large signals:polymer and salt induced (psi)-type circular dichroism [5, 4, 6, 7].While circular dichroism spectroscopy depends on the modulation of incidentlight to detect the differential extinction of circularly polarized light, we haverecently shown that in leaves comparable results can be obtained by measuringthe induced fractional circular polarization of unpolarized incident light [8,9]. As the latter only requires modulation in front of the detector it offersunique possibilities, allowing to probe the molecular architecture from afar. Invegetation, the influence of photosynthesis functioning and vegetation physiologyon the polarizance could provide valuable information in Earth remote sensingapplications, as was demonstrated for decaying leaves [8]. As homochiralityis a prerequisite for these signals (left- and right-handed molecules display anexactly opposite signal and will thus cancel out each other if present in equal2/25umbers) and is unique to nature, circular polarization could also indicate theunambiguous presence of life beyond Earth and as such is a potentially verypowerful biosignature [10, 11, 12, 3, 13, 1].Higher plants evolved relatively recently in contrast to microbial life. Biosig-natures of microbial life are mostly focused on in astrobiology (and which alsodisplay typical circular polarization signals [10]). While molecular analysis sug-gests higher plants appeared by 700 Ma [14], the earliest fossil records date backto the middle Ordovician ( ∼
470 Ma) [15]. The earliest microbial fossil recordsdate back to 3.7 Ga [16] and oxygenic photosynthesis (in cyanobacteria) is likelyto have evolved before 2.95 Ga [17]. It is however unclear if photosyntheticmicrobial life would be able to colonize terrestrial niches extensively enough tobe used as a remotely detectable biosignature.On the other hand, these photosynthetic bacteria stood at the basis of theevolution of higher plants as their photosynthetic apparatus evolved from aendosymbiosis between a cyanobacterium and a heterotrophic host cell. It iswidely accepted that all chloroplasts stem from a single primary endosymbioticevent [18, 19, 20]. Not all photosynthetic eukaryotes, however, descend from thisendosymbiotic host, as certain algae acquired photosynthesis through secondaryendosymbiosis of a photosynthetic eukaryote [20, 21]. The simplified evolutionaryrelations between the different algae, based on the host and on the chloroplasts,are shown in Figure 1.
Figure 1.
Evolutionary relationships based on the host rRNA (left) and basedon chloroplast DNA (cpDNA) (right)Although algae contribute up to 40 % of the global photosynthesis [22], theyhave received limited attention in astrobiology so far. While not as ancient asmicrobial life, algae are considerably older than plants, with fossil evidence ofred algae dating back to 1.6 Ga [23]. Additionally, molecular research on algaehas mainly focused on a few unicellular algae, rather than multicellular species,and systematic studies on the chiral macro-organization of algal photosynthesisare lacking [4]. Despite the common origin, millions of years of evolution hascaused chloroplasts to show a remarkable diversity and flexibility in terms ofstructure.In higher plants the chloroplasts typically display cylindrical grana stacks of10–20 membrane layers that have a diameter of 300 nm – 600 nm. The stacks areinterconnected by lamellae of several hundred nm in length [24]. Additionally,3/25ertain plants can display grana stacks of more than 100 membrane layers [25, 26]while the bundle sheath cells of certain C4 plants, such as maize, lack stackedgrana and only contain unstacked stroma lamellae [27].In higher plants, the psi-type circular polarizance is largely dependent on thesize of the macrodomains formed by the photosystem II light-harvesting complexII supercomplexes (PSII-LHCII). The structure of PSII-LHCII in higher plantsis relatively well known and consists of a dimeric PSII core complex ( C ) andassociated trimeric LHCII, subdivided in three types based on their position andassociation with the core: Loose (L), Moderate (M) and Strong (S). Additionally,three minor antennae occur as monomers (CP24, CP26, CP29) [28]. The positionof trimer L is still unclear and has so far only been observed in spinach [28].The protein constituents and their typical circular polarization signature havebeen determined by T´oth et al ([29]). Furthermore, the negative band of thepsi-type split signal is associated with the stacking of the thylakoid membranes,whereas the positive band is associated with the lateral organization of the chiraldomains [30, 31, 32].The evolutionary history of grana and their functional advantage has been amatter of debate. It has been proposed that the structural segregation by grana ofPSII and PSI prevents excitation transfer between these systems [33, 34, 35]. Theextended compartmentation brought upon by grana might also aid regulatorypathways such as used in carbon fixation [36]. It has been suggested thatgrana facilitate the regulation of light harvesting and enhance PSII functioningfrom limiting to saturating light levels, while at the same time protecting itfrom sustained high irradiance [36]. Together with other adaptations, it hasbeen hypothesized that these changes might have ultimately enabled greenalgae/plants to colonize and dominate various terrestrial niches [34]. Others havesuggested that it might simply be a lack of competition; red algae for instancehave probably experienced several evolutionary bottlenecks, vastly decreasingtheir genome size and therewith their potential for evolutionary adaptation [37].Most closely related to higher plants are the green algae, which share a quiterecent common ancestor. Similar to higher plants, green algae contain chloro-phyll a and b . The structural composition of their photosynthetic machineryand the associated genes are primarily known from the unicellular green algae Chlamydomonas . Despite the high sequence similarity there are significant differ-ences between the supercomplexes of higher plants and green algae. Importantly,green algae lack CP24, resulting in a different organization of the PSII-LHCIIsupercomplex [38]. While many green algae display thylakoid stacking, whichcan be up to 7 membrane layers thick [39], true grana in green algae are rareand only occur in the late branching taxa Coleochaetales and Charales [40, 41].Red algae also contain thylakoid membranes but these are never stacked.Furthermore, unlike green algae and plants, red algae can contain chlorophyll d ,a pigment with an absorption band from 700 nm to 730 nm [42]. The red algaealso contain phycobilisomes that serve as the primary antennae for PSII ratherthan the chlorophyll binding proteins found in higher plants and other algae.These phycobilisomes are homologous to those in cyanobacteria, but are lackingin plants and other algae [20]. 4/25imilarly, brown algae do not possess stacked thylakoid membranes but alsodo not contain phycobilins. All brown algae contain chlorophyll a and usuallychlorophyll c , c and/or c . The light-harvesting systems in brown algae arebased on fucoxanthin chlorophyll a/c , , proteins (FCP), which are homologousto LHC in higher plants/green algae but have a different pigment compositionand organization [43, 44]. Although this is still under debate [45], the brown algaehave been classified as one supergroup [46]. Most brown algae have chloroplastswhich were acquired through one or more endosymbiotic events with red algae[46]. Additionally, certain species of brown algae have been shown to displaypsi-type circular polarizance, although varying magnitudes of these signals havebeen reported, ranging from very weak to signals similar to higher plants (see[4] and references therein).In the present study we measure the fractional circular polarizance of varioushigher plants and multicellular algae. As the level of chiral macro-organizationvaries greatly between unicellular algae, we expect especially in multicellularalgae that the organization can reach a higher or different level of complexity.These studies will additionally assess the feasibility of biosignature detection for(eukaryotic) photosynthesis from different evolutionary stages. While transmis-sion and reflectance generally show a comparable spectral profile, the signalsin reflectance are often weaker (e.g. due to surface glint). In the present studywe will therefore only display the results in transmission, as it provides bettersensitivity for small spectral changes between samples. 5/25 P26 CP29CP24CP26CP29CP24
Trimer STrimer M Trimer S Trimer MPS-II
PS-IATP syn.FCPPS-IIPhycocyaninPhycoerythrin AllophycocyaninPS-IPS-II
CP26 CP29CP26CP29
Trimer LTrimer MTrimer SPS-IITrimer S Trimer MTrimer L
Higher Plants Green AlgaeRed Algae Brown Algae
LHCPS-II PS-I ATP syn.
Figure 2.
Schematic representation of the photosynthetic structures of higherplants and algae. There is a distinct organizational difference in the super-complexes between higher plants and algae. Additionally, while green algaedisplay stacked thylakoid membranes, they lack true grana. Red algae containphycobilisomes, unlike the other algae. In brown algae the thylakoid membranesare threefold and the supercomplex organization is not entirely resolved. 6/25
Materials and Methods
Ulva lactuca , Porphyra sp. and
Saccharina latissima were grown in April atthe Royal Netherlands Institute for Sea Research (NIOZ), using natural lightand seawater. The algae were transported and stored in seawater at roomtemperature. Measurements on the algae were carried out within 2 days afteracquisition.
Ulva sp. , Undaria pinnatifida , Grateloupia turuturu , Saccharina latissima , Fucus serratus and
Fucus spiralis were collected by Guido Krijger from WildWier from the North Sea near Middelburg in February. The algae were transportedunder refrigeration and stored in seawater. Measurements on the algae werecarried out within 2 days after acquisition.Leaves of Skimmia japonica and
Prunus laurocerasus were collected in Januaryfrom a private backyard garden near the city centre of Amsterdam,
Aspidistraelatior was obtained from the Hortus Botanicus Vrije Universiteit Amsterdamin February.
For all measurements, three different samples were used (n=3) and each singlemeasurement is the average of at least 20000 repetitions. Before each mea-surement the samples were padded with paper towels to remove excess surfacewater. Circular polarization measurements were carried out in transmissionand were performed using TreePol. TreePol is a dedicated spectropolarimetricinstrument developed by the Astronomical Instrumentation Group at the LeidenObservatory (Leiden University). The instrument was specifically developed tomeasure the fractional circular polarization (
V /I ) of a sample interacting withunpolarized light as a function of wavelength (400 nm to 900 nm) and is capableof fast measurements with a sensitivity of ∼ ∗ − . TreePol applies spectralmultiplexing with the implementation of a dual fiber-fed spectrometer usingferro-liquid-crystal (FLC) modulation synchronized with fast read-out of theone-dimensional detector in each spectrograph, in combination with a dual-beamapproach in which a polarizing beam splitter feeds the two spectrographs withorthogonally polarized light (see also [8]).In this study we have measured the induced fractional circular polarizance nor-malized by the total total transmitted light intensity ( V /I ). Circular dichroismmeasures the differential absorption of left- or right-handed circularly polarizedincident light, which is often reported in degrees θ . Under certain conditions,these two can be related and can therefore be converted by V /I ≈ πθ deg (see also[3]). It has been shown that for leaves in transmission the induced polarizance Any mention of commercial products or companies within this paper is for informationonly; it does not imply recommendation or endorsement by the authors or their affiliatedinstitutions.
Figure 3.
Circular polarimetric spectra of
Skimmia japonica , Prunus laurocera-sus and
Aspidistra elatior leaves. Shaded areas denote the standard error, n=3per species.The circular polarization spectra of three different higher plants are shown inFigure 3. For all species we observe the typical split signal around the chlorophyll a absorption band ( ≈
680 nm) with a negative band at ≈
660 nm and a positiveband at ≈
690 nm. The spectra of
Skimmia and
Prunus are very similar to eachother in both shape and magnitude and show no significant differences. Theseresults are also very similar to the results obtained for most other higher plants(data not shown). Interestingly, the circular polarimetric spectrum of
Aspidistraelatior shows an exceedingly large negative band ( − . ∗ − ) with a noticeablenegative circular polarization extending much further into the blue, beyond thechlorophyll a (but also b ) absorption bands. The positive band, however, has asimilar magnitude (+6 ∗ − ) as the other two plant species. The circular polarization spectra of two different green algae are shown in Figure4. Similar to higher plants a split signal is observed around the chlorophyll8/25 igure 4.
Circular polarimetric spectra of
Ulva lactuca and
Ulva sp. greenalgae. Shaded areas denote the standard error, n=3 per species. a absorption band ( ≈
680 nm). Unlike higher plants, however, the negativeand positive band do not seem to overlap. The negative band reaches a
V /I minimum at ≈
655 nm and the positive band reaches a maximum at ≈
690 nm,but the
V /I signal is close to 0, and thus shows no net circular polarizationbetween ≈
665 nm to 678 nm. Additionally, the magnitude of the signals ismuch smaller than that of higher plants.
We show the circular polarization spectra of two different red algae in Figure5. These spectra show distinct differences compared to the higher plants andthe green or brown algae.
Porphyra sp. shows a continuous split signal around ≈
680 nm, and an additional sharp positive feature at ≈
635 nm.
Grateloupiaturuturu lacks these features and shows an inverse split signal around ≈
680 nm.In both species, non-zero circular polarization can also be observed between 550nm to 600 nm. We will further interpret these results in the Discussion. 9/25 igure 5.
Circular polarimetric spectra of
Porphyra sp. and
Grateloupiaturuturu red algae. Shaded areas denote the standard error, n=3 per species.
The brown algae exhibit a lot of variation in signal strength. For ease ofcomparison, the results of our circular spectropolarimetric measurements areplotted in Figures 6 and 7 on the same y-scale. Figure 6 makes clear thata juvenile
Saccharina latissima barely displays a significant signal with theexception of a very weak negative feature (
V /I = − ∗ − ). The mature Saccharina latissima samples show somewhat stronger bands, although thesignal is still relatively small ( − ∗ − , +1 ∗ − ). The polarimetric spectra ofthe brown algae Undaria pinnatifida , displays a larger signal comparable to thatof higher vegetation.Interestingly, the polarimetric spectra of the brown algae of the genus
Fucus displays very large circular polarization signals, see Figure 7. The alga
Fucusspiralis has a
V /I minimum and maximum of − ∗ − and +2 ∗ − respectively.Additionally, the bands are relatively narrow, with less polarization outside ofthe chlorophyll a absorbance band. In the polarimetric spectra of Fucus spiralis ,and to a lesser extent also of
Undaria pinnatifida , a small negative band can beobserved at 720 nm. Additionally, in the spectra of both
Fucus serratus and10/25 igure 6.
Circular polarimetric spectra of
Saccharina latissima (juvenile andmature) and
Undaria pinnatifida brown algae. Shaded areas denote the standarderror, n=3 per species.
Fucus spiralis a positive band can be observed at 595 nm.
V /I versus absorbance
The
V /I maxima and minima versus the absorbance are shown in Figure 8.A slight correlation is visible between the maximum and minimum magnitudeof the
V /I bands within 650 nm to 700 nm and the absorbance over 675 nmto 685 nm. In general, the magnitude of the bands increases with increasingabsorbance. Both
Fucus serratus and
Fucus spiralis show positive and negativebands with a very large magnitude well outside this trend. This is similar for thelarge negative band of
Aspidistra elatior . On the other hand, mature
Saccharinalatissima and
Porphyra sp. have a relatively low circular polarizance. 11/25 igure 7.
Circular polarimetric spectra of
Fucus serratus and
Fucus spiralis brown algae. Shaded areas denote the standard error, n=3 per species. 12/25 rateloupia turuturuSaccharina latissima juv. Ulva lactucaUndaria pinnatifidaPorphyra sp.Saccharina latissima mat.Ulva sp.Aspidistra elatiorFucus spiralisSkimmia japonicaPrunus laurocerasusFucus serratus
Figure 8.
Maximum extend of the V/I bands within 650 nm - 700 nm againstthe absorbance over 675 nm - 685 nm. Error bars denote the standard error forn=3 per species 13/25
Discussion
Different eukaryotic phototrophic organisms display different circular polarizationspectra, with signal magnitudes that can vary by two orders of magnitude.Chlorophyll a itself exhibits a very weak intrinsic circular polarizance around680 nm[4]. Excitonic coupling between chlorophylls leads to a much largersignal in phototrophic bacteria and certain algae. In many more developedphototrophic organisms the polarization spectra are dominated by the densityand handedness of the supramolecular structures (psi-type circular dichroism),although these signals are superimposed on each other. Thus for identicalchlorophyll concentrations the polarimetric spectral characteristics can vastlydiffer depending on the organization (see also Figure 9). Excitonic PSI-typeIntrinsic
Figure 9.
The three major sources of circular polarizance around the chloro-phyll absorbance band in the red for higher plants for identical chlorophyllconcentrations. Adapted after [4].The typical psi-type circular spectropolarimetric signals observed in vege-tation are the result of the superposition of two relatively independent signalsresulting from different chiral macrodomains in the chloroplast [47, 48, 49, 6].These psi-type bands of opposite sign do not have the same spectral shape andthus do not cancel each other out completely. The negative band is predominantlyassociated with the stacking of the thylakoid membranes, whereas the positiveband mainly derives from the lateral organization of the chiral macrodomainsformed by the PSII-LHCII complexes [32, 50, 51, 6].Plant chloroplasts generally show little variation in structure [52], which14/25s noticeable in the circular polarization spectra of most plants (e.g. see thespectra of
Skimmia and
Prunus in Figure 3). It has been reported before thatthe cpDNA sequences are extraordinarily conserved among plants and nearlyidentical in ferns, gymnosperms and angiosperms [53]. Of course, certain plantscontain more chloroplasts per cell, or contain chloroplasts which are significantlylarger or smaller, but in both cases the normalized circular polarization willremain the same.The polarimetric spectra of
Aspidistra (Figure 3) show a remarkably intensenegative band, unlike the results usually encountered in plants. The positive band,however, has a magnitude that can be expected based on the lower absorbanceas compared to the other higher plants we measured (see also Figure 8). It hasbeen shown that the contribution of both the negative and the positive band isdependent on the alignment of the chloroplasts [6, 48], which might locally bealigned in such a way that only a single band dominates (e.g. near the veins ofleaves [9]). The polarimetric spectra of
Aspidistra , however, can be very wellexplained by the unusually large grana. Previous electron microscopy researchon
Aspidistra elatior chloroplasts revealed grana containing a vast number ofthylakoid layers that may well exceed 100 [26]. As the positive and the negativebands overlap (leading to the split signal), it is to be expected that also thepositive band is larger than encountered normally.Similar to higher plants, also green algae contain PSII-LHCII supercomplexesutilized in photosynthesis. Between green algae and higher plants there are slightdifferences in the trimeric LHCII proteins and their isoforms, and, in addition,the green algae lack one of three minor monomeric LHCII polypeptides (CP24)(see also[54] and references therein). The green algae we measured show a spectralpolarimetric profile that appears very similar to that of plants. However, thenegative band centered around 650 nm is likely an excitonic band resulting fromshort-range interactions of the chlorophylls and the negative, usually stronger,psi-type band around 675 nm is virtually absent. The positive psi-type centeredaround 690 nm, on the other hand, is still present.These results are unlike those reported for the unicellular green algae
Chlamy-domonas reinhardtii , which display a negative excitonic and a negative psi-typeband of equal strength (e.g. see [55]). Importantly, the PSII-LHCII supercom-plexes are far less stable in green algae as compared to plants, and it has beenindicated that the L trimer (as well as the M and S trimers) could dissociate easilyfrom PSII [38]. It has been shown that in
Ulva flattening of the chloroplastsoccurs under illumination, which additionally results in a decrease in thicknessof the thylakoid membrane itself [56]. Such fundamental changes in molecularstructure might easily lead to (partial) dissociation of trimer L, which in turncan lead to the observed apparent absence of the negative psi-type band.The red algae contain a more primitive photosynthetic apparatus that rep-resents a transition between cyanobacteria and the chloroplasts of other algaeand plants. This is also very evident from the displayed spectra in Figure 5.For both species the magnitude of the signal is small and comparable, eventhough
Porphyra sp. had a much larger absorbance (Figure 8), but the spectralshape suggest very fundamental differences in molecular structure. Surprisingly,15/25 orphyra sp. shows a circular polarization spectrum with bands that mightbe associated with psi-type circular polarizance (at 675 nm (-) and at 690 nm(+)). The origin and significance of these signals, however, requires furtherinvestigation. The circular polarimetric spectra of
Grateloupia turuturu lackthese features but show two bands that can be associated with the excitoniccircular polarization bands similar to those in cyanobacteria (at 670 nm (+)and at 685 nm (-)) (cf. [10]), which for a large part result from the excitonicinteractions in PSI [57]. In both species, the features between 550 nm and 600nm might be associated with R-phycoerythrin [58]. Additionally, in
Porphyra sp. the sharp feature around 635 nm can be associated with phycocyanin [10]. Bothpigment-protein complexes belong to the phycobilisomes, which only occur inred algae and cyanobacteria and function as light harvesting antennae for PSIIwhile LHC is limited to PSI.As in red algae and green algae, the brown algae contain no true grana butthe thylakoid membranes are stacked in groups of three [59]. The brown algaemeasured in this study additionally contain chlorophyll c , which is slightly blue-shifted compared to chlorophyll a or b . Compared to chlorophyll a , chlorophyll c however has only a very weak contribution to the overall circular polarizance.Additionally, in brown algae the light-harvesting antennae are homogeneouslydistributed along the thylakoid membranes [60, 61].Interestingly, the juvenile Saccharina displays only a very weak negativeband around 683 nm (Figure 6). These results closely resemble those of isolatedbrown algae LHCs, which exhibit no excitonic bands but show solely a negativeband around 680 nm. This band likely results from an intrinsic induced chiralityof the chlorophyll a protein complex [61]. The polarimetric spectra of mature Saccharina and
Undaria show a split signal that is similar to that of higherplants. While the molecular architecture of the LHCs is very different from thosein higher plants, the pigment-protein complexes in brown algae are organized inlarge chiral domains which give similar psi-type signals in circular polarizance[62, 63]. These intrinsic so-called fucoxanthin chlorophyll a/c binding proteinsshow a high homology to LHC in higher plants and have been shown to formcomplexes with trimers or higher oligomers [64, 65, 66].As shown in Figure 7, the measured species of the genus
Fucus exhibit anunusually large signal in circular polarizance, while the absorbance of the sampleswas within the range of the samples of the other species (Figure 8). Althoughtheir spectral shapes are very similar to those of diatoms (cf. [67, 62, 61]) thebands are 2 orders of magnitude stronger in
Fucus . Most research on chlorophyll a/c photosynthesis is, however, carried out on diatoms and the reported size ofthe protein complexes again varies. Signals of such magnitude suggest that thesemacromolecular assemblies are much larger in
Fucus than previously reportedfor other brown algae. Additionally, in the spectra of
Fucus a positive bandcan be observed around 595 nm. Most likely, this band and the weaker negativeband around 625 nm can be assigned to chlorophyll c .The results here show that the molecular and macromolecular organizationof the photosynthetic machinery in algae is much more flexible and dynamicthan reported before, likely due to larger inter-specific differences than generally16/25ssumed. Additionally, this also appears to be the case for one of the plants wemeasured ( Aspidistra elatior ), which displayed a negative psi-band one order ofmagnitude larger than ordinarily observed for higher plants.When it comes to circular polarizance as a biosignature, it is important tonote that efficient photosynthesis is not necessarily accompanied by large signalsin circular polarization. While the intrinsic circular polarizance of chlorophyllis very low, the magnitude of the signals become greatly enhanced by a largerorganization resulting in excitonic circular polarizance and ultimately psi-typecircular polarizance. For the latter, the chiral organization of the macrodomainsof the pigment-protein complexes is of importance, but it should be noted thatthe density of the complexes needs to be large enough (that is, significant couplingover the macrodomain is required) in order to function as a chiral macrodomain[5]. Many organisms thus display only excitonic circular polarizance, as is thecase for certain algae measured in this study and generally bacteria. Whenpsi-type circular polarizance is possible, the signals can easily become very large,in our study up to 2 % for brown algae in transmission.
We have measured the polarizance of various multicellular algae representingdifferent evolutionary stages of eukaryotic photosynthesis. We have shownthat the chiral organization of the macrodomains can vary greatly betweenthese species. Future studies using molecular techniques to further characterizeand isolate the complexes in these organisms are highly recommended. It willadditionally prove very interesting to investigate these chloroplasts (includingthose with larger grana such as
Aspidistra) using polarization microscopy (e.g.[68, 49, 69]). The high quality spectra in this study and their reproducibilityunderline the possibility of utilizing polarization spectroscopy as a quantitativetool for non-destructively probing the molecular architecture in vivo .Our results not only show variations in spectral shapes, but also in magnitude.Especially the brown algae show a large variation, which is up to three ordersof magnitude for the species measured in this study. Additionally, the inducedfractional circular polarization by members of the genus
Fucus is much larger thanobserved in vegetation. Future studies on the supramolecular organization in thisgenus and the variability caused by, for instance, light conditions, will also clarifythe maximum extent of the circular polarizance by oxygenic photosyntheticorganisms.While the displayed results were obtained in transmission, the spectralfeatures are also present in reflection. As such, future use of circular spec-tropolarimetry in satellite or airborne remote sensing could aid in detecting thepresence of floating multicellular algae but also aid in species differentiation,which is important in regional biogeochemistry [70].Importantly, while the presence of similar circular polarization signals isan unambiguous indicator for the presence of life, life might also flourish on aplanetary surface and still show minimal circular polarizance (which for instancewould have been the case on Earth if terrestrial vegetation evolved through17/25ifferent Archaeplastida/SAR supergroup lineages). On the other hand, thesesignals might also be much larger than we would observe from an Earth disk-averaged spectrum (which is the unresolved and therefore spatially integratedspectrum of a planet).
We thank Klaas Timmermans (NIOZ) and Guido Krijger (Wildwier) for provid-ing us with the algae samples. We thank the Hortus Botanicus Vrije UniversiteitAmsterdam for providing us with the Aspidistra samples. This work was sup-ported by the Planetary and Exoplanetary Science Programme (PEPSci), grant648.001.004, of the Netherlands Organisation for Scientific Research (NWO).18/25 eferences
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