Transformation of Thylakoid Membranes during Differentiation from Vegetative … · Transformation of Thylakoid Membranes during Differentiation from Vegetative Cell into Heterocyst

Transformation of Thylakoid Membranes duringDifferentiation from Vegetative Cell into HeterocystVisualized by Microscopic Spectral Imaging1[W]

Shigeichi Kumazaki*, Masashi Akari, and Makoto Hasegawa

Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606–8502,Japan (S.K., M.A., M.H.); and Precursory Research for Embryonic Science and Technology, Japan Science andTechnology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan (S.K.)

Some filamentous cyanobacteria carry out oxygenic photosynthesis in vegetative cells and nitrogen fixation in specialized cellsknown as heterocysts. Thylakoid membranes in vegetative cells contain photosystem I (PSI) and PSII, while those in heterocystscontain predominantly PSI. Therefore, the thylakoid membranes change drastically when differentiating from a vegetative cellinto a heterocyst. The dynamics of these changes have not been sufficiently characterized in situ. Here, we used time-lapsefluorescence microspectroscopy to analyze cells of Anabaena variabilis under nitrogen deprivation at approximately 295 K. PSIIdegraded simultaneously with allophycocyanin, which forms the core of the light-harvesting phycobilisome. The otherphycobilisome subunits that absorbed shorter wavelengths persisted for a few tens of hours in the heterocysts. The whole-thylakoid average concentration of PSI was similar in heterocysts and nearby vegetative cells. PSI was best quantified byselective excitation at a physiological temperature (approximately 295 K) under 785-nm continuous-wave laser irradiation,and detection of higher energy shifted fluorescence around 730 nm. Polar distribution of thylakoid membranes in theheterocyst was confirmed by PSI-rich fluorescence imaging. The findings and methodology used in this work increased ourunderstanding of how photosynthetic molecular machinery is transformed to adapt to different nutrient environments andprovided details of the energetic requirements for diazotrophic growth.

The most essential pigment-protein complexes foroxygenic photosynthesis are PSI and PSII, which areembedded in the thylakoid membranes of chloroplastsand cyanobacteria. Cooperation between PSI and PSIIachieves light-driven noncyclic electron transport fromthe oxidative splitting of water to the reduction offerredoxin and is accompanied by the generation ofa proton gradient for ATP synthesis. Phycobilisomes(PBS), another pigment-protein complex, are attachedto the stromal side of the thylakoid membrane in cya-nobacteria and red algae; they work as light-harvestingantennae to transfer electronic excitation energy mainlyto PSII and, in some cases, to PSI (Gantt 1994). The in-tegration of these pigment-protein complexes changesin response to light conditions, nutrient status, anddevelopmental stage (Fujita et al., 1994; Grossman et al.,1994; Wolk et al., 1994).

Some cyanobacteria, including Anabaena variabilis,are able to grow diazotrophically using the nitrogen-

fixing enzyme nitrogenase. Because nitrogenase is sen-sitive to oxygen, oxygenic photosynthesis is not readilycompatible with diazotrophic growth. When thisfilamentous cyanobacterium is grown under fixednitrogen-deficient conditions, approximately 1 in 10 to20 vegetative cells differentiates into a heterocyst, inwhich oxygenic photosynthesis is suppressed and ni-trogenase becomes operative (Haselkorn, 1978; Wolket al., 1994). The other vegetative cells continue oxygenicphotosynthesis. The differentiation of heterocysts fromchains of vegetative cells has been studied extensively(Golden and Yoon, 2003; Toyoshima et al., 2010). Theabundances of PSII and PBS decrease during the transi-tion. PSI appears to persist in the heterocyst to produceATP by cyclic electron transport, because nitrogen fix-ation demands a large amount of ATP (Wolk et al.,1994). However, the mechanisms by which PBS andPSII are degraded during heterocyst differentiationremain unclear, and whether the amount of PSI per cellchanges is unknown.

The PBS of A. variabilis contain three types of phyco-biliproteins, pigment-protein complexes with distinct ab-sorption and fluorescence spectra. The core PBS containsallophycocyanin (APC), which absorbs around 654 nm(Ying and Xie, 1998); the core is most closely connected toPSII. More peripherally in the PBS, the so-called rodcontains phycoerythrocyanin (PEC) and phycocyanin(PC), which absorb maximally around 575 and 604 to 620nm, respectively (Switalski and Sauer, 1984; Zhang et al.,1998). Photon energy is absorbed by PEC, then transferred

1 This work was supported in part by the Ministry of Education,Culture, Sports, Science, and Technology of Japan (grant no. 19056012to S.K.).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Shigeichi Kumazaki ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.112.206680

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downhill through PC and APC and finally to PSII. Thestructure of PBS is probably optimized not only forefficient energy transfer to PSII and/or PSI but also fortransformation and/or degradation under variousnutrient conditions. However, the order in which thesesubunits degrade during heterocyst differentiation re-mains unknown. One strategy to address this questionis to isolate heterocysts at several stages during differ-entiation and quantify their proteomes via mass spec-trometry. However, such isolation procedures workwell only when there is a good understanding of theproperties of cells at different stages. Ideally, noninva-sive methods should be used to understand changes inthe integrity of PSII and PBS in intact cells in filaments.

In principle, time-lapse microscopic observationscan clarify the process of differentiation from a vege-tative cell into a mature heterocyst. Spectral micros-copy is an ideal tool to analyze physiological stateand/or amounts of pigment-protein complexes undervarious conditions. Acquiring microscopic fluorescencespectra of individual cells is a natural extension of laserscanning confocal fluorescence microscopy, which hasbeen applied to several types of cyanobacterial cells,including heterocysts (Peterson et al., 1981; Ying et al.,2002; Wolf and Schüssler, 2005; Kumazaki et al., 2007;Vermaas et al., 2008; Sukenik et al., 2009; Bordowitz andMontgomery, 2010; Collins et al., 2012, Sugiura andItoh, 2012). Microscopic fluorescence spectra reflect theconcentration of pigment-protein complexes and theenergy transfer dynamics between photosynthetic pig-ments. However, to date, there have been no thoroughtime-lapse investigations of the fluorescence spectra ofheterocysts and vegetative cells during the differentiationprocess.

In this study, we investigated the dynamic changesin thylakoid membranes of A. variabilis during het-erocyst differentiation. Our unique microscopic systemcan acquire fluorescence spectra from an entire linearlyilluminated region with about 2-nm wavelength reso-lution in a single exposure (Kumazaki et al., 2007).Heterocyst formation was induced by transferringvegetative cell filaments from fixed-nitrogen-sufficientincubation medium to nitrogen-deprived medium. Weconducted long-term observations (60–96 h) on identicalfilaments. Another unique feature of our setup is that ituses a near-infrared (NIR) excitation laser source. Ourprevious microspectroscopic study of chloroplasts of ahigher plant, maize (Zea mays), and a green alga (Para-chlorella kessleri) showed that continuous wave (CW)laser light emitting at 785 to 820 nm excited PSI withhigh selectivity under the one-photon excitation (OPE)mode. This enabled us to observe highly PSI-rich flu-orescence spectra and images with signals around 710to 740 nm, even at approximately 295 K (Hasegawaet al., 2010, 2011). We used this technique to quantifyPSI in individual heterocysts compared with its parentaland contiguous vegetative cells. Pigment fluorescenceunder OPE qualitatively differed from that under two-photon excitation (TPE) using a pulsed NIR laser (typi-cally achieved with picosecond or femtosecond pulses),

because TPE using 800 to 830 nm resulted in spectrawith contributions from PBS, PSII, and PSI, as typicallyobserved by visible light excitation (Kumazaki et al.,2007; Hasegawa et al., 2010, 2011). The advantages ofour microscopic system are the high wavelength res-olution and coverage of the entire fluorescence spec-trum, the availability of fluorescence spectra at severaldifferentiation stages, and the multiple excitation modeswith different selectivities for pigment-protein complexes.Together, these analyses allowed us to characterizespectral decomposition and to understand the time de-pendence of different pigment-protein complexes, even ata physiological temperature. Microscopic absorptionspectra were also obtained from single cells. Thesedata were tentatively used to estimate the absoluteconcentrations of PSI and PSII in heterocysts andvegetative cells.

RESULTS

Time-Lapse Fluorescence Spectral Imaging by TPE andTransmission Spectral Imaging

A time course of fluorescence images of a single fil-ament under 808-nm TPE at five selected fluorescenceregions was generated with our microscopic fluores-cence spectrometer (Fig. 1). The five regions were F615(600–630 nm), F643 (630–655 nm), F662 (650–670 nm),F685 (670–700 nm), and F715 (700–730 nm), which ap-proximately corresponded to PEC, PC, APC, PSII, andPSI fluorescence regions, respectively, as confirmed byour spectral analyses (Supplemental Fig. S1). After 2 hof nitrogen deprivation, all cells had similar shapes inthe bright-field images and similar intensities in thefluorescence images. After 24 h of nitrogen deprivation,the terminal cell in the filament appeared to be largerthan the other cells (Fig. 1; T620, T680, and T730), atypical feature of heterocysts. The terminal cell alsofluoresced more weakly than the other cells, especiallyat F662, F685, and F716, although the signal at F643was comparable to that of the other cells. From 24 to 96h, all fluorescence signals of the terminal cell graduallydecreased to zero, except at F715, where it retained afaint signal.

Bright-field microscopy images with monochro-matic illumination reflect absorption by photosyn-thetic pigments at the selected wavelengths of 615 to625 nm (Fig. 1; T620), 675 to 685 nm (T680), and 725 to735 nm (T730). At T620, the terminal cell became moretransparent (brighter) over time than the nearest cell,indicating a decrease in abundance of phycobilinmolecules due to degradation of the PBS complex, asreported previously (Grossman et al., 1994; Wolk et al.,1994). The difference in transmission between the ter-minal cell and its neighbor was less obvious at T680than at T620; at 96 h, the darkness of the terminal cellwas similar to that of the nearest vegetative cell, indi-cating the presence of chlorophylls. Thus, the T620 andT680 images indicated a substantial decrease in PBS inthe terminal heterocyst, but the amount of chlorophyll

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remained high compared with the nearest vegetativecell. At T730, there appeared to be almost no differencein brightness, and hence transmission, between theterminal cell and its neighbor, because photosyntheticpigments absorb light only very weakly at 730 nm.More detail was obtained by plotting absorption

spectra for individual cells (Fig. 2). The vegetative cellsalso showed subtle, but reproducible, changes in ab-sorption. Absorption around 600 to 680 nm, which isprimarily attributable to PBS and red light absorptionby chlorophylls, decreased around 24 h after nitrogendeprivation, then slightly increased until 96 h. We alsoanalyzed absorption spectra in individual filamentcells that were grown for longer than 10 d in nitrogen-free BG-11 medium (Fig. 2D) and found that theirspectral features were similar to those of the heterocystand vegetative cells at 96 h.The fluorescence spectra of heterocyst cells clearly

changed over time, while there were only small changesin those of vegetative cells (Fig. 3). At 2 h, two majorpeaks, at 663 6 1.5 and 684 6 1.5 nm, were observed inboth preheterocysts and vegetative cells. In the hetero-cyst at 24 h, the overall intensity of the fluorescencedecreased, and the main peak was located around 648nm, which was clearly blue shifted compared with thepeak at 2 h (Fig. 3A). Between 24 and 97 h, there was alarge decay at wavelengths less than 700 nm, while thefluorescence intensity remained nearly constant around730 nm. In vegetative cells, the main decay in intensitywas at around 620 nm, and there were minor decays atwavelengths less than 660 nm (Fig. 3, B and C). Therewas also a small increase in intensity at around 685 nm.

Time-Lapse Fluorescence Spectral Imagingby NIR OPE

The fluorescence images obtained under 785-nmOPE (Fig. 4) differed in several respects from those

obtained under 808-nm TPE (Fig. 1). From the earliest(2 h) to the latest (75 h) observation time, the fluores-cence intensities in the F615 and F643 regions were tooweak for detailed analyses of individual cells. Theseweak intensities were explained by preferential excita-tion of relatively long-wavelength-absorbing pigments(Hasegawa et al., 2010, 2011). The F715 intensities ofheterocyst cells remained comparable to those of vege-tative cells up to 72 h. The F662 and F685 intensitiesdecayed in the heterocyst cell, but the rates of decaywere slower than those observed under TPE (Fig. 1).These relatively slow decay rates of APC and PSIIcorrelated with the delayed change in cell shape ob-served in the bright-field images (T620, T680, and T730in Fig. 4). The terminal cell was already larger than thenearby vegetative cells at 24 h (Fig. 1; T620, T680, andT730), while differences in cell size and shape first be-came visible at 48 h when fluorescence images wereobtained with 785-nm OPE (Fig. 4). The different tim-ings of heterocyst formation did not appear to be due todifferent excitation laser conditions but to factors notyet completely controlled (e.g. extracellular residualnitrogen and/or intracellular stock of fixed nitrogen), aswill be addressed below with examples.

Overall, the microscopic fluorescence spectra ofheterocysts under 785-nm OPE were very similar tothose of adjacent vegetative cells (Fig. 5, A and B).There were small but reproducible differences betweenthe heterocyst and vegetative cells in the wavelengthregion around 650 to 690 nm, while the spectral shapesaround 715 to 755 nm were nearly identical (Fig. 5C).These fluorescence spectra were also very similar tothose of heterocyst cells under 808-nm TPE (Fig. 5C).Notably, the spectra containing PSI fluorescence as thedominant contribution were reminiscent of the fluo-rescence spectrum at 77 K of A. variabilis vegetative cellsunder nitrogen-sufficient growth conditions (Petersonet al., 1981; Mannan and Pakrasi, 1993), which showed

Figure 1. Time-lapse microspectroscopic imagesof A. variabilis filaments, including a terminal celldifferentiating into a heterocyst, under 808-nmTPE. Fluorescence images were taken at wave-length regions 600 to 630 nm (F615), 630 to 655nm (F643), 655 to 670 nm (F662), 670 to 700 nm(F685), and 700 to 730 nm (F715). Bright-fieldimages with monochromatic illumination weretaken at 615 to 625 nm (T620), 675 to 685 nm(T680), and 725 to 735 nm (T730). Each gray-scale fluorescence image was scaled by its max-imum and minimum intensities except in thebottom row (at 96 h), which was scaled by 40% ofthe maximum intensity to visualize weakly fluo-rescent heterocysts. White and black arrows in-dicate terminal cells. Bar = 10 mm.

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peaks at 666, 685, 695, and 735 nm (Fig. 5D). The fluo-rescence spectra measured at 295 K appeared to reflectspectral broadening of the low-temperature spectrum.

Spectral Decomposition of Single-CellFluorescence Spectra

We obtained 1,625 fluorescence spectra from indivi-dual cells at various stages of heterocyst differentiation

using two different excitation methods (785-nm OPEor 808-nm TPE). Simultaneous analysis of all thesespectra by singular value decomposition (SVD) yieldedfive component spectra (Supplemental Figs. S1 and S2;Supplemental Materials and Methods S1). The fivecomponents, PEC, PC, APC, PSII, and PSI, were suf-ficient and necessary to fit all raw single-cell spectra,including different stages of heterocyst differentiation(Fig. 6). Analyses of spectral decomposition yieldedtime-dependent amplitudes of the five spectral com-ponents (Figs. 7 and 8). Under 808-nm TPE (Fig. 7), theAPC and PSII components were very similar to eachother in heterocyst dynamics (Supplemental Text S2);compared with the other components in heterocystand vegetative cells, both decayed quickly and com-pletely to zero in developing heterocysts but remainedlargely unchanged in neighboring vegetative cells. ThePC components in heterocyst cells increased tran-siently before finally decaying to a lower value thanthe original intensity, while vegetative cells showed a

Figure 2. Time-lapse observations of microscopic absorption spectra ofsingle heterocysts and neighboring vegetative cells of A. variabilis. Eachspectrum is the average of four individual cells from separate filaments,including the one shown in Figure 1, at equivalent times and filamentpositions. A, Heterocysts (Het.). B, Vegetative cells adjacent to heterocysts(Veg. 1). C, Vegetative cells adjacent to Veg. 1 (Veg. 2). Spectra in A to Cwere obtained through rigorous time-lapse experiments over 96 h. D,Averaged microscopic absorption spectra of heterocysts (solid line) andvegetative cells (broken line) from filaments grown diazotrophically forlonger than 10 d in a shaken flask and transferred to a glass-bottomed dishjust before microscopic measurements. Error bars show SD of 37 spectra forheterocysts and 182 spectra for vegetative cells. Absorption spectra werenot normalized when comparing absorbance among different cells/times.

Figure 3. Time-lapse observations of microscopic fluorescence spectraof single heterocysts and neighboring vegetative cells of A. variabilisexcited with 808-nm TPE. Each spectrum is the average of seven in-dividual cells from separate filaments, including the one shown inFigure 1, at equivalent times and filament positions. A, Heterocysts(Het.). B, Vegetative cells adjacent to heterocysts (Veg. 1). C, Vegeta-tive cells adjacent to Veg. 1 (Veg. 2). Fluorescence spectra were notnormalized when comparing relative intensities among different cells/times. a.u., Arbitrary units.


Kumazaki et al.






gradual and monotonic decrease. The PEC componentin heterocysts decayed to below the original intensity,but the decay rate was much slower than that of APC.In vegetative cells, the PEC component decreased rap-idly in the first 40 h and remained at approximately10% to 15% of the value recorded at 2 h. The PSIcomponent in heterocysts remained comparable to, orslightly higher than, the original value at 2 h, while itdecreased slightly in vegetative cells.The response times of the fluorescence spectra to

nitrogen deprivation were similar or well synchronizedamong different filaments in the same glass-bottomeddish, but an independent experiment on filaments innominally identical culture conditions showed a tem-poral delay (Supplemental Fig. S3; compare with Fig. 7or its normalized version, Supplemental Fig. S4). Asidefrom the delay in responses, the relative timing ofchanges in the different spectral components was similarbetween the two independent experiments. For instance,in both data sets, the peak in PC fluorescence intensitywas recorded when the APC and PSII fluorescences firstreached about 20% to 30% of their original intensities.In the decomposition data obtained under 785-nm

OPE (Fig. 8; Supplemental Fig. S5), PSI fluorescence wasthe dominant component, and its intensity remainedessentially unchanged in both heterocysts and vegetativecells. The APC component had decayed to zero atapproximately 72 h in the heterocysts, but the PSIIcomponent in heterocysts decayed more slowly. Thisdifference in decay rate should be carefully inter-preted, as discussed below.Time-lapse analyses of identical filaments undergoing

heterocyst differentiation (Figs. 7 and 8; SupplementalFigs. S3–S5) were obtained by illuminating several iden-tical filaments with lasers at different time points; dataat a single time point consisted of lateral scans (xyscans) at three different focal depths (z sections). There-fore, the cumulative effects of photochemical damagemayhave affected heterocyst differentiation. To examine suchartifacts, several glass-bottomed dishes containing A.variabilis filaments were prepared from the same cellsuspension at a time defined as 0 h. Several filamentsin one of the dishes were microspectroscopically

analyzed once at a single time point after fixed-nitrogen starvation began. The time-dependent inten-sities of the spectral components in these experiments(Supplemental Figs. S6 and S7) were nearly identical tothose in the rigorous time-lapse experiments (Figs. 7and 8; Supplemental Figs. S3–S5). These data in-dicated that repeated laser illumination had little ef-fect on heterocyst formation and that differentiation of A.variabilis filaments was well synchronized even amongdifferent glass-bottomed dishes under these experi-mental conditions. We also sometimes observed celldivisions in filaments that were repeatedly analyzedby spectral imaging at 10- to 24-h intervals. Therefore,cellular viability appeared to have been preserved underthese experimental conditions.

Heterocyst and Vegetative Cells under Long-TermDiazotrophic Growth Conditions

We obtained microscopic spectral features of A. vari-abilis filaments grown under diazotrophic conditions forlonger than 10 d in a shaken flask. The filaments weretransferred to a glass-bottomed dish just before micro-scopic experiments (Fig. 2D; Table I; Supplemental Figs.S8 and S9). PSI fluorescence intensity remained largelyidentical between the heterocyst and vegetative cells inthe same filaments under both 785-nm OPE and 808-nmTPE; the PSI signal observed under 785-nm OPE wasparticularly stable (Supplemental Fig. S9E). PEC intensitywas also very low in both heterocyst and vegetative cells(Supplemental Figs. S8A and S9A). At 2 h in the time-lapse measurements under 808-nm TPE, relative peakintensities of PEC in both the heterocysts and vegetativecells averaged 10% to 20% of the APC component invegetative cells (Table I; Fig. 7; Supplemental Figs. S3and S4), but on average, less than 1% of relative peakintensity was detected in both heterocyst and vegetativecells in cultures grown diazotrophically for more than10 d. These findings indicated that PEC was stably pre-sent only when exogenous fixed nitrogen was supplied,at least under these growth conditions. Some heterocystsappeared to contain PC, APC, and PSII componentsduring long-term diazotrophic growth (Supplemental

Figure 4. Time-lapse microspectroscopic imagesof A. variabilis filaments, including a terminal celldifferentiating into a heterocyst, under 785-nmOPE. Panels are as described in Figure 1. Bar =10 mm.



















Figs. S7 and S8). However, because we could not de-termine the age of heterocysts in the long-term diazo-trophic culture, some of them may have been immatureand therefore still contained detectable amounts of PC,APC, and/or PSII.

Intracellular Distribution of Thylakoid Membranes inthe Heterocyst

There was a nonhomogenous distribution of PSI inthe fluorescence images of both intercalary and terminal

heterocysts under 785-nm OPE (Fig. 9; SupplementalFig. S10). PSI fluorescence inside the heterocysts oftenshowed a gradient parallel to the filament axis, asreported previously from an electron microscopy study(Maldener and Muro-Pastor, 2010). The intercalaryheterocysts often showed bright PSI fluorescence peaksnear their connections to adjacent vegetative cells, whiletheir central regions were relatively dark. This sharpwithin-cell fluorescence contrast was not observed in

Figure 6. Spectral decomposition of microscopic fluorescence spectrain Figure 3. Experimental spectra are shown with black circles,whereas spectral components are shown as follows: PEC is a blue solidline (peak at 620 nm), PC is green (closed squares; peak at 646 nm),APC is magenta (peak at 661 nm), PSII is brown (white circles; peak at686 nm), and PSI is a black broken line (peak around 730 nm). Sim-ulated spectra are shown with black solid lines. For details, see textand Supplemental Materials and Methods S1. a.u., Arbitrary units;Het., heterocyst; Veg. 1, vegetative cell adjacent to the heterocyst.

Figure 5. Time-lapse observations of microscopic fluorescence spectra ofsingle heterocysts and neighboring vegetative cells of A. variabilis under785-nm OPE. A and B, Spectra are averages of seven individual cells fromseparate filaments, including the one shown in Figure 4, at equivalent timesand filament positions. A, Heterocysts (Het.). B, Single vegetative cellsadjacent to heterocysts (Veg. 1). C, Comparison of three single-cell fluo-rescence spectra rich in PSI. Het., TPE represents averaged spectra of het-erocysts under 808-nm TPE, Het., OPE represents averaged spectra ofheterocysts under 785-nm OPE, and Veg., OPE represents averaged spectraof single vegetative cells under 785-nm OPE. These three spectra wereselected for their smaller contributions from PSII and PBS compared withother spectra obtained under equivalent conditions. D, Fluorescencespectrum of vegetative cells of A. variabilis grown photoautotrophically andmeasured at 77 K (from Mannan and Pakrasi, 1993). a.u., Arbitrary units.


Kumazaki et al.







vegetative cells under 785-nm OPE. Vegetative cells hadrelatively low-fluorescence regions near their centersbecause thylakoid membranes are localized at the pe-riphery of the cytoplasm (Maldener and Muro-Pastor,2010), as confirmed by the central dark regions of veg-etative cells under 808-nm TPE (Fig. 1). The spatial res-olution under 785-nm OPE (only slit confocal effect) wasworse than under 808-nm TPE (slit confocal effect andsuppressed out-of-focus excitation), especially in depth(Hasegawa et al., 2010). Thus, the central regions ofvegetative cells (Fig. 9, B and C) appeared to fluorescestrongly because of the peripheral thylakoid membranesbelow and above the true three-dimensional center of thecells.

DISCUSSION

Different Dynamic Changes of PBS Subunits and PSIIbetween Heterocyst and Vegetative Cells

The following descriptions of PBS subunits and PSII,particularly PEC and PC, focus mainly on fluorescencedata obtained under 808-nm TPE. In the time-lapsemicroscopic fluorescence spectra, the decomposition

patterns clearly indicated that the decay of APC fluo-rescence was highly synchronized with that of PSII(Fig. 7; Supplemental Figs. S3, S4, and S6). AlthoughPEC, PC, and APC are subunits of the PBS, PC and PECfluorescences decayed much later than that of APC dur-ing heterocyst formation. In fact, the PC and PEC fluo-rescences rose and then decayed at around the time thefluorescence intensities of APC and PSII reached 20%to 30% of their original values. In one experiment, thedecay in PSII and APC fluorescence amplitudes did notstart within the first approximately 15 h (SupplementalFig. S3), which is later than in an equivalent experiment(Fig. 7; Supplemental Fig. S6). The difference may re-flect residual fixed nitrogen in the culture solution and/or within cells. However, the relative timing of changesamong the five fluorescence components was wellpreserved among independent experiments.

Greater pigment fluorescence can generally be at-tributed to increased concentrations and/or a decreaseof nonradiative decay processes, including energytransfers. The decrease in absorbance around 600 to640 nm, where PC subunits absorb, in heterocysts waslargely monotonic (Fig. 2A; Ducret et al., 1996);therefore, the transient increase in PC fluorescenceis best explained by a decrease in energy transfer

Figure 8. Time dependence of five flu-orescence components obtained fromeight filaments with heterocysts of A.variabilis in the same glass-bottomeddish under 785-nm OPE. Average am-plitudes (solid lines) are shown withoutnormalization. Error bars show SD. Bro-ken lines showmaximum and minimumvalues. a.u., Arbitrary units; Het., het-erocyst; Veg. 1, vegetative cell adjacentto heterocyst.

Figure 7. Time dependence of fivefluorescence components obtainedfrom seven filaments with heterocystsof A. variabilis in the same glass-bot-tomed dish under 808-nm TPE. Aver-age amplitudes (solid lines) are shownwithout normalization. Error bars showSD. Broken lines show maximum andminimum values. a.u., Arbitrary units;Het., heterocyst; Veg. 1, vegetative celladjacent to heterocyst.









efficiency from PC to APC. The energy acceptor, APC,was more rapidly lost from the original PBS complexthan were the PC and PEC energy donors. The fast andsynchronous degradations of APC and PSII seemed tobe physiologically critical to suppress energy transferto PSII and consequent oxygen evolution, which isessential for rapid nitrogenase functionality. The syn-chronous behavior of APC and PSII observed here wasconsistent with the recently reported statistical corre-lation analysis of fluorescence spectra of 313 Nostocpunctiforme cells, including heterocysts, at 40 K basedon an excitation wavelength of 532 nm (Sugiura andItoh, 2012). In that analysis, the stoichiometric ratiobetween APC and PSII (APC/PSII) was more stablethan the ratios of other pairs of fluorescence compo-nents, although no time-lapse measurement was per-formed and APC/PSII was more scattered and loweron average in heterocysts than in vegetative cells.Notably, PSII fluorescence appeared to decay moreslowly during heterocyst differentiation than that ofAPC under 785-nm OPE (Fig. 8; Supplemental Fig. S5).Direct excitation of PSII by 780-nm OPE was reported(Thapper et al., 2009). Previously, we showed that ex-citation of PSII in chloroplasts was detectable under785-nm OPE in plant mesophyll cells (maize) and agreen alga (P. kessleri; Hasegawa et al., 2010, 2011). Inthose studies, the contribution of PSII to total fluores-cence was more substantial than that observed in thiswork on vegetative cells of A. variabilis. Thus, directexcitation of PSII and/or indirect excitation of PSIIthrough PSI (uphill spillover) at physiological temper-atures is probably observable and larger than the exci-tation of APC under 785-nm OPE. Therefore, 785-nmOPE, and not 808-nm TPE, appeared to indicate thatvery small but detectable amounts of PSII remained fora longer time in heterocysts.

Several studies have reported that remaining PBS inheterocysts work as light-harvesting antennae for PSI

(Peterson et al., 1981; Cardona et al., 2009). The rela-tively long lifetime of the rod part of PBS (PEC and PC)compared with the core (APC) observed in heterocystsin this work was consistent with the previous findingthat heterocysts in Anabaena sp. 7119 accumulate PBSwithout APC (Yamanaka and Glazer, 1983). Two typesof PBS with different linker proteins (CpcG1 andCpcG2) were found in the unicellular cyanobacteriumSynechocystis sp. PCC 6803 (Kondo et al., 2007). Onewas a normal PBS consisting of a PC rod and APC core(CpcG1-PBS); the other was an abnormal PBS lackingAPC (CpcG2-PBS) that seemed to transfer excitationenergy preferentially to PSI (Kondo et al., 2007). The

Figure 9. Heterocyst of A. variabilis on a glass-bottomed dish showingpolar distribution of PSI-rich fluorescence after 96 h of nitrogen dep-rivation. A, Bright-field image using a conventional spectrum of illu-mination without a narrow band-path filter. B, Fluorescence imageobtained under OPE (CW of 785 nm) laser irradiation. The integratedfluorescence wavelength range was 700 to 753 nm. C, Fluorescenceintensity (Flu. Int.) profile of the data in B calculated along the lineindicated by the arrowheads. a.u., Arbitrary units.

Table I. Peak amplitudes of fluorescence components in heterocysts and adjacent vegetative cells of A. variabilis

Excitation Mode Cell TypeTime after Fixed-Nitrogen

DeprivationPEC PC APC PSII PSI

TPE (808 nm, 0.2 picoseconds,76 MHz)

Heterocyst 2 ha 0.20 0.14 1.01 0.90 0.1596 ha 0.007 0.034 0.006 0.015 0.18

.10 db 0.003 0.012 0.08 0.08 0.10Vegetative cell adjacent to

heterocyst2 ha 0.21 0.135 1c 0.96 0.15

97 ha 0.015 0.023 1c 1.23 0.10.10 db 0.003 0.098 1c 0.52 0.12

OPE (785 nm, CW) Heterocyst 2 hd n.d.e n.d.e 0.99 0.80 17.072 hd n.d.e n.d.e n.d.e 0.32 16.7

.10 df n.d.e n.d.e n.d.e 0.1 14.0Vegetative cell adjacent to

heterocyst2 hd n.d.e n.d.e 1c 0.88 17.4

72 hd n.d.e n.d.e 1c 1.46 16.4.10 df n.d.e n.d.e 1c 1.10 14.5

aAverage values from Supplemental Figure S4, derived from Figure 7. bAverage values from Supplemental Figure S8. cAll amplitudes weredivided by that of APC of the vegetative cell adjacent to the heterocyst in a filament. dAverage values from Figure 8. eUnder 785-nm OPE,normalized amplitudes below 0.1 were designated not detected (n.d.). Note that the absolute signal intensity of APC of the vegetative cell adjacent tothe heterocyst under 785-nm OPE was about 2 orders of magnitude smaller than that under 808-nm TPE; therefore, a value of 0.1 under 785-nm OPEwas comparable to 0.001 under TPE. fAverage values from Supplemental Figure S9.


Kumazaki et al.







rod part of PBS in A. variabilis heterocysts analyzed inthis work may be similar to CpsG2-PBS in Synecho-cystis sp. PCC 6803. Given the very low-level fluores-cence of PEC in heterocysts relative to that of PC underlong-term diazotrophic conditions (more than 10 d;Table I), the PSI light-harvesting antennae in heterocystsseemed to contain mainly PC and a reduced contentof PEC compared with PBS rods in fixed-nitrogen-supplemented culture. Some decay of PC and PECfluorescences over a few tens of hours in heterocystsmay be caused not only by proteolytic degradation butalso by newly formed energy transfer coupling to PSI.Cardona et al. (2008) reported that the vegetative cell

adjacent to the heterocyst showed transient differencesin fluorescence properties compared with other vegeta-tive cells in N. punctiforme. Our spectral decompositionresults, however, showed no reproducible differences influorescence between the nearest and second-nearestvegetative cells to heterocysts (Supplemental Figs. S3–S7). The difference between our results and theirs may bedue to species differences and/or our lower samplingfrequency (only once or twice per day).The effects of nitrogen starvation on PBS in non-

diazotrophic cyanobacteria were previously studied inSynechococcus sp. PCC 6301 (Yamanaka and Glazer,1980) and sp. PCC 7942 (Collier and Grossman, 1992).In those studies, PBS degradation began with thetrimming of peripheral PC rods, accompanied bydegradation of the APC core. The monotonic decreaseof PEC in vegetative cells of A. variabilis was similar toperipheral rod trimming (Fig. 7; Supplemental Figs. S3,S4, and S6). There was no recovery of PEC fluorescencein vegetative cells even after the heterocyst begansupplying fixed nitrogen after approximately 24 h. Onthe other hand, PC and/or APC in PBS seemed to in-crease in vegetative cells after approximately 24 h, asevidenced by increases in absorbance in the 600- to650-nm region (Fig. 2, B and C). PC fluorescence invegetative cells decreased more slowly than that ofPEC, and in some cases it even increased slightly(Supplemental Fig. S6). Notably, fluorescence intensitiesof APC and PSII were somehow maintained in vege-tative cells, even 60 to 96 h after the onset of nitrogendeprivation diminished almost all PEC (Fig. 7;Supplemental Figs. S3, S4, and S6). Thus, some minorportion of PEC may have been originally uncoupledwith PSII in terms of energy transfer, and degradationof this highly fluorescent PEC was one of the earliestresponses to nitrogen starvation, allowing rapid recyclingof amino acids from the pigment-protein complexes.All our microscopic measurements were performed

at 295 6 2 K (approximately 22°C), lower than thepreviously reported optimum temperature for Ana-baena spp. growth of around 28°C to 30°C (Wang et al.,2007). Despite the temperature difference, the appear-ance of a transient minimum of microscopic absorptionat 24 h (Fig. 2) appeared to match the changes in PCand chlorophyll contents per cell 24 h after nitrogendeprivation reported in cells at approximately 303 K(Ehira and Ohmori, 2006). The timing of the transient

minimum indicated that degradation of PBS and PSIIapparently stopped at around 24 h, and nitrogensupplied by the heterocysts enabled vegetative cells toincrease photosynthetic pigments. PBS fluorescence inheterocysts increased after fixed-nitrogen deprivation,and its intensity peaked at around 20 h in Anabaenaspp. at 32°C (Toyoshima et al., 2010). This timing wasvery similar to the PC fluorescence peak (approximately24 h) in our time-lapse measurements under TPE (Fig.7), which was acquired from the same filaments asanalyzed for microscopic absorption spectra (Fig. 2).Given these comparisons of the transient features ofthylakoid membranes, there appeared to be no signifi-cant differences in heterocyst differentiation dynamicsbetween 22°C and 28°C to 32°C. The timing of fluo-rescence changes was similar among all our data setswhen the lag phase before the start of APC fluorescencedecay was taken into account (Supplemental Fig. S3).

Many studies have examined the timing and spatialpatterns of gene expression after the onset of nitrogendeprivation in heterocyst-forming cyanobacteria (Floresand Herrero, 2010; Kumar et al., 2010; Maldener andMuro-Pastor, 2010; Christman et al., 2011; Flahertyet al., 2011). For example, heterocyst differentiation wascritically controlled by two regulator proteins, HetR andNtcA, and their encoding genes were strongly expressedin certain spatially separated cells that ultimately becameheterocysts (Black et al., 1993; Olmedo-Verd et al., 2006).Our laboratory is currently investigating the timing andspatial relationships between the transformation of thy-lakoid membranes unveiled in this work and the ex-pression of genes related to the transition to diazotrophicgrowth conditions.

Number and Oligomeric Status of PSI and PSII inSingle Cells

The fluorescence intensities shown as spectra andamplitudes in our figures were averaged on a per-pixelbasis across whole-cytoplasm areas enclosed by con-tour lines of fluorescent regions. This means that av-erage per-pixel fluorescence signals were equivalent toaverages over whole thylakoids in single cells. Themost direct estimate of PSI concentration in individualcells, at least in our work, was given by the micro-scopic fluorescence spectra under 785-nm OPE. Therewas no substantial difference in the whole-thylakoidPSI concentration between heterocysts and adjacentvegetative cells from the initiation of heterocyst dif-ferentiation to heterocyst maturity (Figs. 4, 5, A and B,and 8; Supplemental Figs. S5 and S7). Because of thedecrease in PSII and PBS in heterocysts, the relativeabundance of PSI should inevitably increase. This pre-diction was confirmed by a recent mass spectrometry-based proteomic study on filaments of A. variabilis (Owet al., 2009). In that study, the relative abundance of PSIwas 3.3-fold higher on average in heterocyst cells thanin vegetative cells. If proteomic analysis is conductedon samples with an adjusted concentration of total













proteins, the original absolute concentrations of pro-teins in intact single cells may be difficult to estimate.Our work is unique in that the concentration of PSI perthylakoid in single cells was directly compared be-tween heterocysts and their parent or adjacent vege-tative cells. Our method of quantifying PSI involvedlittle interference from possible energy transfer be-tween PBS and PSI, because of the direct excitation ofPSI by the CWNIR laser and the relatively small uphillenergy transfer from PSI to the other pigment-proteincomplexes.

The total quantity of PSI in single cells was esti-mated by integrating the fluorescence between 700 and753 nm under 785-nm OPE across the whole cell(Supplemental Text S3). We estimated that the averagequantity of PSI per cell was comparable between in-tercalary heterocysts and single vegetative cells butthat a single terminal heterocyst contained approxi-mately 30% less PSI than a single vegetative cell(Supplemental Table S2). These estimates contradictedthe results of a previous study on Anabaena cylindrica, inwhich the quantity of PSI was 1.5 times higher in het-erocysts than in vegetative cells (Alberte et al., 1980).

In the cells of another heterocystous cyanobacte-rium, N. punctiforme, the PSI complex was reported tobe in equilibrium between the monomer and trimer,which had fluorescence peaks around 728 and 750 nm,respectively, at 77 K (Cardona and Magnuson, 2010).However, another study proposed that this spectralchange was attributable to equilibria among themonomer, dimer, and tetramer based on the phylo-genetic similarity between Anabaena sp. PCC 7120 andN. punctiforme (Watanabe et al., 2011). A change in theoligomerization status of the PSI complex was accompa-nied by changes in absorption and fluorescence spectra ofthe long-wavelength-absorbing spectral forms, some ofwhichwere detectable at approximately 295 K (Karapetyanet al., 2006). We previously demonstrated that theoligomeric status of chlorophylls with various fluores-cence peaks could be sensitively detected under 785-nmOPE (Hasegawa et al., 2011). Thus, the oligomeric statusof PSI probably remained largely unchanged in hetero-cysts and vegetative cells during the adaptation to nitro-gen deprivation, because there was an almost constantspectral shape in the wavelength region between 700 and755 nm (Fig. 5).

The absolute concentration of chlorophyll a wasderived from absorbance values of the microscopicabsorption spectra (Fig. 2; Supplemental Table S2). Weestimated average chlorophyll quantities on a whole-cellbasis, although there was clearly an uneven distributionof thylakoid membranes in the cytoplasms of both het-erocysts and vegetative cells (Figs. 1 and 9B; SupplementalFig. S10B). The value for A680 of mature heterocyst wasapproximately 70% of that of vegetative cells when A.variabilis filaments were grown diazotrophically forlonger than 10 d (Fig. 2D). The decline in absorbancewas primarily attributed to a decrease in chlorophyllconcentration in single cells, because even the longestwavelength-absorbing subunit of PBS, APC, showed

only 5% to 7% of absorbance compared with the peak ataround 650 nm (Grabowski and Gantt, 1978; Murakamiet al., 1981; Rolinski et al., 1999; MacColl et al., 2003).Based on these absorbance values and an estimate ofthe optical path length (Supplemental Text S4), the totalamounts of chlorophyll in the terminal and intercalaryheterocysts were estimated to be 50% and 71%, respec-tively, of that in a vegetative cell (Supplemental Table S2).The mass of chlorophyll per cell was estimated as 0.12,0.17, and 0.23 picograms (pg) in terminal heterocysts, in-tercalary heterocysts, and vegetative cells, respectively(Supplemental Table S2). These values were largelyconsistent with those obtained by whole-culture pigmentanalysis and cell counts of A. cylindrica (Alberte et al.,1980; 0.09 pg in heterocysts, 0.17 pg in vegetative cells).

Because all chlorophyll a remaining in heterocysts isprimarily attributable to PSI, the absolute amountof PSI can be estimated in single heterocyst cells(Supplemental Table S2). The different numbers of PSIunits between single terminal (0.88 3 106) and singleintercalary (1.2 3 106) heterocysts seemed to be duenot to the density of PSI on thylakoid membranes butto the different extents of fluorescent area of PSI(Supplemental Table S1), which can sometimes be vi-sualized by the unipolar and bipolar distribution ofPSI in terminal and intercalary heterocysts, respec-tively (Fig. 9B; Supplemental Fig. S10B). The differencemay reflect the number of vegetative cells to whichfixed nitrogen must be transported.

If the decrease in chlorophyll during heterocystdifferentiation is attributed only to the complete dis-appearance of PSII, and if the relatively constant con-centration and oligomeric status of PSI are taken intoaccount, the stoichiometric ratio between PSII and PSIin a vegetative cell can be estimated. Based on a simplemodel and parameters (Supplemental Table S2), thePSII-to-PSI ratio on a monomer basis (PSII/PSI) invegetative cells was estimated as 1.0. This ratio waseither consistent with or slightly larger than previouslyreported values for A. variabilis (PSII/PSI = 0.7–1.4[Kawamura et al., 1979]; approximately 0.3–0.6 [Fujitaet al., 1985]), Synechocystis sp. PCC 6714 (approxi-mately 0.5–0.7; Murakami et al., 1997), Synechocystis sp.PCC 6803 (approximately 0.5–0.8; Hihara et al., 1998),and N. punctiforme (approximately 0.26; Cardonaet al., 2007). These relatively low estimates for PSII/PSI were based on biochemical treatments and anal-yses of whole-cell suspensions. A recent micro-spectroscopic study on Nostoc sp. KU001 showed thatthe absorbance at around 680 nm in the heterocyst wasabout 48% of that in a vegetative cell (Sugiura andItoh, 2012). This value would yield an even greaterPSII/PSI ratio than ours if the amount of PSI weresimilar between heterocysts and vegetative cells in thatspecies. Further examination of the reliability of mi-croscopic absorption spectroscopy to quantify photo-synthetic pigments in living cells will be useful.Verification should include comparisons with othermethods, other samples, and various experimentalconditions.


Kumazaki et al.















CONCLUSION

We characterized the differentiation of A. variabilisvegetative cells into heterocysts and the adaptationdynamics of vegetative cells to diazotrophic growthconditions at approximately 295 K using time-lapsemicrospectroscopy. During heterocyst differentiation,APC and PSII showed a highly synchronous decaywith a constant stoichiometric ratio within approxi-mately 30 h. In all cells, including vegetative ones, PECand PC decayed during the transition from nitrogen-replete to nitrogen-deficient conditions before theheterocysts became operative. These decays slowed orceased after heterocyst function began. The transientincrease in PEC and/or PC fluorescence in (proto)het-erocysts was best explained by the more rapid decays ofAPC and PSII than of PC and PEC. The decompositionof microscopic fluorescence spectra into PEC, PC, APC,PSII, and PSI components was greatly assisted by thehighly pure PSI spectrum obtained at approximately295 K under 785-nm OPE. That spectrum had very littleinterference due to energy transfers from other shorterwavelength-absorbing pigment-protein complexes.Selective excitation of PSI was useful to quantify andimage PSI in cyanobacteria as well as in plant and algachloroplasts (Hasegawa et al., 2010, 2011). The averagewhole-thylakoid concentration of PSI did not sub-stantially change during heterocyst differentiation, in-dicating that the large ATP demand by nitrogenasecould be fully satisfied by cyclic electron flow with theoriginal density of PSI in the thylakoid membranes.The constant spectral shape of PSI fluorescence under785-nm OPE during differentiation was best explainedby the largely unchanged oligomeric status of the PSIcomplex. This combination of microscopic absorptionand fluorescence spectral data obtained under 785-nmOPE is a promising method to quantify PSI and PSII inindividual intact living cells.

MATERIALS AND METHODS

Strain and Growth Conditions

Anabaena variabilis (strain NIES-2095) cells were purchased from the mi-crobial culture collection at the National Institute of Environmental Studies inTsukuba, Japan. Before fixed-nitrogen deprivation, filaments were grownphotoautotrophically in normal BG-11 medium (Stanier et al., 1971) for 10 to20 d at 29°C, which corresponded to the midexponential growth phase underthese conditions. The photosynthetic photon flux density at the sample positionin the incubator was about 20 mmol photons m22 s21. Cell cultures were grownunder a 15-h-light/9-h-dark photoperiod. Nitrogen-free BG-11 medium wasprepared by removing NaNO3 and replacing ferric ammonium citrate with ferriccitrate in the BG-11 formulation. The nitrogen deprivation conditions were asfollows: an aliquot of the cell suspension, typically 1 cm3, was centrifuged for 60s at 1.8 3 103 g. The supernatant (greater than 0.97 cm3) was removed, and thepellet was diluted with nitrogen-free BG-11 medium to a volume of 1.0 cm3. Thisprocess was repeated twice to ensure nitrogen deprivation. The final suspensionwas transferred to a glass-bottomed dish (D111505; Matsunami). The uppersurface of the glass was coated with poly-L-Lys before the suspension of A.variabilis filaments was added to immobilize cells. A grid pattern on the lowerglass surface was used to locate and examine several identical filaments at 12- to24-h intervals for up to 60 to 96 h after the start of nitrogen starvation.

We conducted two types of time-lapse microscopic observations. First,several identical filaments in the same glass-bottomed dish were inoculated

from an original culture suspension and kept continuously on the microscopestage (time-lapse experiment). In this experiment, identical filaments were laserilluminated several times, and we could not rule out the possibility that illu-mination affected the physiological changes. Second, multiple glass-bottomeddishes were simultaneously inoculated from an original culture suspension.Each dish was used once to analyze several filaments at a single observationtime (single-time experiment). Except when being microscopically analyzed,these dishes were continuously illuminated by white fluorescent light (pho-tosynthetic active radiation of approximately 15 mmol photons m22 s21).

Microscopic Spectroscopy and Analysis

Details of the microspectroscopy systems have been described previously(Kumazaki et al., 2007; Hasegawa et al., 2011). The system is based on aninverted microscope with an oil-immersion microscope objective (UPLSAPO,100XO, numerical aperture = 1.40; Olympus). For microscopic fluorescencespectral measurements, the average laser power at the sample position was35 6 5 mW in the case of TPE with a pulsed laser centered at 808 nm. In thecase of OPE of mainly PSI with a CW laser centered at 785 nm, the averagelaser power at the sample position was 30 6 2 mW. The excitation laser wasresonantly scanned at 7.9 kHz along a linear region of approximately 20 mm.Microscopic fluorescence spectra were recorded with an EMCCD camera at-tached to an imaging polychromator. We used an exposure time of 0.5 s perarray of pixels with a length of approximately 20 mm. The microscopic ab-sorption spectrum was given by the ratio in a bright-field image betweenregions with and without a filament of cells in the region of interest. Mono-chromatic illumination (centered at 600, 610, 620, 640, 650, 660, 671, 680, 690,700, and 730 nm, with full-width at half-maximum of 10 nm) was automati-cally changed by a filter wheel on a halogen lamp (Hasegawa et al., 2011).Offset absorbance at a given wavelength was the difference in apparent ab-sorbance between the wavelength and 730 nm. Fluorescence and absorptionspectral imaging were performed at three z positions, with intervals of 0.6 mm.All microscopic measurements were conducted in a laboratory at 295 6 2 K.

Analyses of microscopic data, including SVD of many spectra of individualcells, were performed using a set of in-house programs in Mathematica ver-sion 5.2 (Wolfram Research). Single-cell fluorescence spectra were averages ofsingle-pixel spectra in the intracellular regions enclosed by contour lines alongthe cell periphery given by a human-assisted averaging program, which alsogave the areas of fluorescent regions in individual cells. Single-cell absorptionspectra were calculated as described previously (Hasegawa et al., 2011). Be-cause of focus position drift, some z-section data that were clearly far from thecentral height of cells were excluded from averaging.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Fluorescence spectra of PEC, PC, APC, PSII, andPSI.

Supplemental Figure S2. Five spectra derived by singular value decom-position of 1,635 single-cell spectra.

Supplemental Figure S3. Time dependence of relative peak amplitudes offive normalized spectral components in six A. variabilis filaments, includ-ing heterocysts and adjacent vegetative cells.

Supplemental Figure S4. Normalized amplitudes of data shown in Figure 7.

Supplemental Figure S5. Time dependence of five fluorescence compo-nents obtained from eight A. variabilis filaments with heterocysts in thesame glass-bottomed dish under 808-nm TPE.

Supplemental Figure S6. Time dependence of fluorescence components insingle-time experiments under 808-nm TPE.

Supplemental Figure S7. Time dependence of fluorescence components insingle-time experiments under 785-nm OPE.

Supplemental Figure S8. Cell position dependence of relative spectral am-plitudes of fluorescence components under 808-nm TPE in cells growndiazotrophically for longer than 10 d.

Supplemental Figure S9. Cell position dependence of relative spectral am-plitudes of fluorescence components under 785-nm OPE in cells growndiazotrophically for longer than 10 d.














Supplemental Figure S10. Polar distribution of PSI fluorescence in termi-nal heterocyst.

Supplemental Table S1. Average area of PSI-rich fluorescence regionsunder 785-nm OPE.

Supplemental Table S2. Trial estimation of absolute amounts of PSI andPSII monomer units in single cells.

Supplemental Text S1. Eighty-three singular values obtained in SVD anal-yses of 1,625 single-cell spectra.

Supplemental Text S2. Note on independence and synchronization be-tween APC and PSII.

Supplemental Text S3. Note on whole-cell integrated PSI fluorescence in-tensities.

Supplemental Text S4. Estimation of the absolute quantity of chlorophyll ain single cells.

Supplemental Materials and Methods S1.

ACKNOWLEDGMENTS

We thank Profs. M. Terazima, H. Inoue, Y. Mizutani, M. Fujii, H.Masuhara, S. Itoh, and K. Yoshihara for their encouragement. We thankProfs. H. Oh-oka and M. Nishiyama and Mr. M. Ghoneim, Mr. M. Yabuta,and Mr. K. Miyata for valuable contributions to the preliminary stages ofthis work.

Received September 1, 2012; accepted December 23, 2012; published Decem-ber 28, 2012.

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