Plant Physiol. (1986) 80, 843-8470032-0889/86/80/0843/05/$0 1.00/0

The Effects of Osmotic Tissue Dehydration and Air Drying onMorphology and Energy Transfer in Two Species of Porphyra1

Received for publication September 18, 1985 and in revised form November 18, 1985

CELIA M. SMITH2, KAZUHIKO SATOH3, AND DAVID C. FORK*Carnegie Institution of Washington, Department ofPlant Biology, Stanford, California 94305

ABSTRACT

Studies were conducted to document the effects on morphology andenergy transfer in photosynthesis of severe tissue dehydration inducedeither by air-drying or by immersing the tissues of two Porphyra speciesin hyperosmotic solutions. These studies showed that the dehydration-tolerant intertidal alga, Porphyra perforata J.Ag., was almost unaffectedby either of these treatments, while the dehydration-sensitive Porphyranereocystis Anders. was damaged similary by both treatments. Damageto that sensitive species was characterized by ruptured organelles as seenby interference microscopy as well as by increased fluorescence emissionat 682 nanometers emanating from allophycocyanin. These results sug-gest that a disruption of energy transfer between allophycocyanin andchlorophyll a occurs because of the damage to membranes followingtissue dehydration, and that the increase in the yield of phycobilinfluorescence is a good indicator of these phenomena. Thus, air-dryingand osmotic-dehydration appear to have similar physiological conse-quences in a dehydration-sensitive alga but almost no effect in a tolerantspecies.

Macrophytic marine algae are distributed in distinct zones incoastal regions from intertidal areas that are rarely wetted byincoming waves to subtidal areas that are never exposed by lowtides. The algae in these communities differ in their physiologicaltolerance of air-drying, and these differences explain in part theirobserved zonation (4, 15, 18). Those species adapted to live inthe intertidal region are tolerant of severe dehydration that canbe induced by air-drying (4, 16). Intertidal species are alsotolerant of osmotic dehydration, but subtidal and low tidalspecies are intolerant of both kinds of dehydrations (1, 15, seealso 2).

Because air-drying of algal tissues to a specific water contentis difficult, a new approach was needed to study the basis ofadaptation made by marine algae to dehydration. We consideredthe possibility that immersing algal tissues in hyperosmotic so-lutions may parallel the buildup of salts in the extracellular waterof air-dried algae, and may be an effective means of experimen-tally dehydrating tissues. Providing that algal cells are imperme-able to NaCl, exposure to a solution with increased salt concen-tration withdraws water from cells to a water potential defined

'Carnegie Institution of Washington, Department of Plant Biology,Publication No. 859.

2 Present address: Department ofBotany, National Museum ofNaturalHistory, Smithsonian Institute. Washington DC 20560

3 Present address: Department of Pure and Applied Sciences, Collegeof Arts and Sciences, University of Tokyo, 3-8-1, Komaba, Meguro-ku,Tokyo 153. Japan.

by the concentration of salts in the external medium. Because ofthe lack ofa cuticle, algal tissues rapidly equilibrate with solutionsof different water potentials. Thus, hyperosmotic dehydrationallows greater reproducibility for replicate dehydrations than ispossible with air-drying.

Studies of the characteristics of fluorescence emission in fullyhydrated and in air-dried tissues of Porphyra perforata (5, 1 1)revealed alterations in distributions of light energy upon dehy-dration. In this study, fluoresence emission was used to followchanges in energy transfer resulting from air-dehydration andosmotically induced dehydration in the intertidal species, P.perforata, and the subtidal species, Porphyra nereocystis. No-marski interference micrography was used to compare fine struc-tural details of cells prior to, during, and after dehydration fromboth methods and for both species.

MATERIALS AND METHODS

Plant Material. Porphyra perforata J. Ag. was collected atHopkins Marine Station, Pacific Grove, CA, at a tidal elevationof +0.6 m. The subtidal epiphyte, Porphyra nereocystis Anders.was collected at -6.7 m from submerged thalli of its brown algalhost, Nereocystis lutkeana Post. and Rupr.

Thalli were maintained for no more than 3 d in the laboratoryat 12°C, in open dishes containing seawater. Osmotic treatmentof samples was carried out at 21C in the dark for 13 min byplacing discs punched from thalli in a solution that was over 10-fold more concentrated than seawater, 5.41 M NaCl in seawater(pH 8.20) with 45 mm Hepes. The calculated water potential forthis solution is lower than -10 MPa. Samples were air-dried inthe dark for 1 h in a stream of cool air.

Fluorescence emission spectra were obtained using the micro-processor system described previously (6) and were corrected ona quantum basis using measurements made with a standardlamp. A small disc of tissue was held against the end of the 8mm diameter quartz light guide, which was immersed in liquidN2. Green actinic light, absorbed predominantly by the phycob-ilin pigments, was used to excite fluorescence and was obtainedby passing white light from a 150 W tungsten lamp (type DLS)through two Corning CS 4-96 and CS 3-69 colored glass filtersand a Balzers heat-reflecting filter.

Tissue samples were examined and photographed with No-marski interference microscopy before, during hyperosmotic de-hydration or air-drying, and 1 min after rehydrating tissues innormal seawater.

RESULTSNormarski Interference Microscopy. Light micrographs are

shown in Figure 1 for tissues of both P. perforata and P. nereo-cystis before, during, and 1 min after rehydration after air-dryingor osmotic dehydration. Cells of air-dried tissues appeared re-duced in volume (cJf Fig. 1, A, B, F, G) although internal details

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FIG. 1. Photomicrographs obtainedwith Nomarski optics of tissue samples oftwo Porphyra species: A, fully hydratedtissues of P. perforate; B, tissues while airdried; C, air-dried tissues 1 min after re-hydration in seawater; D, tissue while os-motically dehydrated; E, osmotically de-hydrated tissues I min after rehydrationin seawater; F, fully hydrated tissues of P.nereocystis, G, tissues while air-dried; H,air-dried tissues I min after rehydrationin seawater, I, tissues while osmoticallydehydrated; J, osmotically dehydrated tis-sues 1 min after rehydration in seawater.Techniques for dehydration are describedin "Materials and Methods."

such as the position or boundary of the single chloroplast werenot visible. When seawater was added to air-dried tissues of P.perforata, cell volume increased markedly within 1 min, andorganelles appeared intact (Fig. IC). When seawater was addedto air-dried tissues of P. nereocystis, fine structural detail wasdisrupted, and cellular contents appeared nearly homogeneous(cf. Fig. 1, F, H) indicating a loss of cellular integrity.The question whether osmotically induced dehydration pro-

duces effects similar to those of air-drying was examined bydehydrating tissues of P. perforate and P. nereocystis in a nearlysupersaturated solution of NaCl in seawater (5.41 M NaCI in

seawater). Examination using interference microscopy showedthat osmotic or air dehydration of P. perforate produced similareffects because samples immersed in the hyperosmotic solutionhad reduced cellular volume, and fine cellular detail was notseen (cf. Fig. 1, B, D). Upon rehydration, chloroplast and cellvolume of P. perforate returned to normal (Fig. 1, A, E), as wasobserved for air-dried samples of P. perforate (Fig. IC).Osmotic or air dehydration of P. nereocystis produced similar

effects because osmotically dehydrated tissue had reduced cellu-lar volume, and lacked fine cellular detail (cf. Fig. 1, G, I). Afterrehydration, P. nereocystis had ruptured chloroplasts, and its

-

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EFFECTS OF DRYING ON TWO SPECIES OF PORPHYRA

cells lacked fine structural detail (Fig. I J) as did air-dried samplesof P. nereocystis (Fig. lH).

Fluorescence Emission Spectra of Two Species of Porphyra.Fluorescence emission spectra are shown in Figure 2 for P.perforata before (A) and after (B) osmotic dehydration. We canassign the shoulders and maxima seen in Figure 2 as follows: 647nm, PC4; 660 nm, APC II/III; 685 nm, both to APC-B (7) andChl a of PSII; 695 nm, Chl a associated with PSII; and 730 nm,Chl a associated with PSI. The spectrum of Figure 2B shows thatthe fluorescence of phycobilins were decreased somewhat (in-cluding the 685 nm band), suggesting that osmotic dehydrationgave rise to more efficient energy transfer among phycobilinpigments and between phycobilins and Chl a. Fluorescence ofthe Chl bands (695 and 730 nm) was little affected by dehydra-tion, indicating that the connection of PBsomes to thylakoidmembranes remained largely intact, and that the efficiency ofenergy transfer of the green light absorbed by phycobilins to Chla was largely unaffected.

After 15 min of rehydration in seawater, no further significantchange in the fluorescence emission spectrum for the osmoticallydehydrated tissues of P. perforata were seen (Fig. 3B). The veryminor alteration in fluorescence emission suggests that P. perfor-ata is tolerant of severe dehydration more negative that -10

73012

10 7

695

a, 685

VD A0234 BFL ~ i

64721660I_650 700 750 800

Wavelength, nmFIG. 2. Fluorescence emission spectra at 77 K in P. perforata before

(A) and after (B) osmotically inducing tissue dehydration. Techniquesfor dehydration and measurements of fluorescence spectra are describedin "Materials and Methods."

73012-

10 7695

8

4-

2

650 700 750 800Wavelength.nm

FIG. 3. Fluorescence emission spectra at 77 K in P. perforata beforeosmotically inducing tissue dehydration (A) and after 15 min of rehydra-tion in seawater (B).

4 Abbreviations: PC, phycocyanin; APC, allophycocyanin; APC-B, thelong wavelength form of all allophycocyanin; PBsome, phycobilisome.

MPa average tissue water potential.The question whether osmotically induced dehydration pro-

duces effects similar to those induced by air dehydration in P.nereocystis was examined by immersing tissues in a solution ofthe same concentration used to dehydrate P. perforata. Afterosmotic dehydration, no apparent alteration of energy transferwas seen as long as the tissues of P. nereocystis were maintainedin the dehydrated state (Fig. 4B). This result suggests that dehy-dration itselfdid not damage the photosynthetic apparatus. Dam-age induced by osmotic dehydration was seen as a large increaseof fluorescence at 682 nm (primarily from APC-B), and asincreased fluorescence ofother phycobilins in rehydrated samples(Fig. 4C). These results indicate that osmotic dehydration of P.nereocystis produces not only a loss in cellular fine structure, butalso a major disruption of energy transfer between APC and Chla. The increased fluorescence of other phycobilin pigments alsoindicates that energy transfer between phycobilins and Chl a hasbeen reduced. It is clear from these results that P. nereocystis isnot intolerant of severe dehydration.

Fluorescence emission spectra are shown in Figure 5 for P.nereocystis before (A), during (B), and after (C) air-drying ofthalli. Changes in the fluorescence emission spectra ofrehydratedtissues of this subtidal species were again observed (cf. Fig. 5, A,C). Upon rehydration, phycocyanin fluorescence at 662 nm wasincreased, and fluorescence at 695 nm (PSII) was decreased (Fig.5B) when compared with fully hydrated tissues (Fig. 5A). A largefluorescence band at 682 nm, originating from APC-B, andincreases in fluorescence from other phycobilin bands were alsoseen.

Air-drying P. perfiorata under the same conditions used to dryP. nereocystis produced a decrease in fluorescence emission fromPSII bands at 685 and 695 nm as seen by Oquist and Fork (11)(data not shown). After rewetting, the samples had the samefluorescence emission spectra as shown in Figure 2A. These datasuggest that air-drying did not produce damage to the photo-chemical apparatus of P. perforata.

12 - 728

10

8-

6956 -

685

CD4 -!2>,

2 -'IIs .

o 728, ,

658-

() 2

0)-

650 700 750 800

Wavelength, nm.FIG. 4. Fluorescence emission spectra at 77 K in P. nereocystis before

osmoticallv inducing- tissue dehvdratinn (A) while dephydrated (B), and15 min after rehydration in seawater (C).

845

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Plant Physiol. Vol. 80, 1986

(D 662

,^2

CD

0 - 68264 ----(D)

028 - t

6-

648

2 -

0

650 700 750 800

Wavelength, nm.

FIG. 5. Fluorescence emission spectra at 77 K in P. nereocystis beforeair drying (A), while dehydrated (B), and 15 min after rehydration inseawater (C).

DISCUSSION

Algae inhabiting the intertidal zone can lose between 80 to90% of their original weight during diurnal low tides (4, 16), andyet fully recover photosynthetic abilities when rehydrated (4).Cells in such dehydrated tissues are probably surrounded bynearly saturated solutions of NaCl plus the other less abundantsalts in seawater. It seems likely that the tolerance of algae in theintertidal region to dehydration can be separated into two parts:(a) the ability to tolerate water loss, and (b) the ability to tolerateincreased salt concentrations around cells. We have seen herethat the sensitivities to dehydration of two closely related speciescorrelated well with the levels of air-drying that are routinelyexperienced in their respective habitats. The apparent physiolog-ical tolerance and stability of its photochemical system afterexposure to high external salt concentrations would seem tomake P. perforata well suited to withstand repeated cycles ofdrying during daily low tides. In contrast, subtidal P. nereocystisis almost never exposed to air-drying, and is damaged similarlyby osmotic dehydration or air-drying.Another study using osmotic dehydration as an experimental

tool for testing photosynthetic tolerance of 12 intertidal andsubtidal algal species found that the distributions of plants werewell correlated with abilities of those species to recover photo-synthesis following osmotic dehydration (CM Smith and JABerry, unpublished results). Moreover, Satoh et al. (14) extendedthe use of osmotic dehydration by investigating effects of inter-mediate levels of dehydration on primary processes of photosyn-thesis of P. perforata. As is shown here, osmotic dehydrationmay be employed to reproducibly dehydrate algal tissues, sincethis treatment produces effects on cell integrity and photosyn-thesis similar to those produced by air-drying.

Although many intertidal species (3-5, 11, 15, 18) reestablish

efficient energy transfer and 02 evolution upon rehydration, thephysiological mechanisms responsible for this tolerance are un-known. One possible explanation may involve protecting effectsof intracellular osmotica acting as compatible solutes (13; seeMunns et aL [10] for a recent review). Work by Reed et al. (12)and Wiencke and Lauchli (17) shows that changes in concentra-tions of two intracellular galactosylglycerols, floridoside and iso-floridoside, occur with changes in external water potentials whenspecies ofPorphyra are cultured in continuously elevated osmoticconditions. These results indicate that floridoside and iso-flori-doside are osmotically active, and suggest that these glycerolsmay be important components of the mechanism of toleranceto daily lowered water potentials imposed by air-drying duringlow tides.One difference, however, between any long-term culture stud-

ies and this work with plants collected from tidal habitats is thepattern of dehydration experienced by these algae prior to study.During exposures at low tide the tissues of intertidal algae dehy-drate and rehydrate on a regime regulated in part by tides andwater vapor condensing on thalli exposed before dawn (16). Thedecrease in cell volume that accompanies tissue dehydration inP. perforata may passively concentrate intracellular osmotica tolevels effective in protecting sensitive cellular components duringdiurnal exposures at low tides. Upon rewetting of tissues follow-ing such exposures, a recovery to the ismotic state with sea-water occurs within 24 h. Thus, it seems unlikely that withinsuch short times there would be a de novo synthesis of osmoticain cells to meet osmotic disruptions. Kremer (8) demonstrated alack of synthesis of glycerols in P. perforata upon air-drying,supporting the concept of a passive increase in osmotica as animportant component of tolerance to air-drying by intertidalspecies. Decreases in water potential of cells could also increasehydrophobic attractions among proteins of phycobilin pigments,which may confer protective effects of PBsomes in tolerantspecies. This idea is supported by the observation of a slightlyincreased efficiency of energy transfer among pigments of dehy-drated P. perforata (Fig. 4B).

Dehydration of P. nereocystis apparently results in substantialmembrane damage since there was a loss of fine structure in cellsof this alga upon rehydration. The observed sensitivity of P.nereocystis to dehydration may relate to different types of os-motica present in the cells, different membrane characteristics,or lessened abilities to regulate inorganic ion concentrationsextracellularly during periods of dehydration.

If membrane damage results from dehydration then a disrup-tion of the ionic environment of an entire cell could be expected.Such a disruption would help explain why energy transfer fromthe PBsome to Chl a of PSII was interrupted in rehydrated P.nereocystis. The maintenance of a proper ionic environment iscritical to efficient energy transfer in PBsome-containing algae(9). Depletion of Ca from cells of the blue-green alga Anacystisnidulans induced an increase in fluorescence emission of phy-cobilins similar to that observed with P. nereocystis (9). Further-more, if Ca was supplied within a certain critical time, thennormal fluorescence emission characteristics were recovered andpermanent damage was avoided. It is possible to suppose that analteration in the membrane structure caused a detachment ofPBsomes from thylakoid membranes. In any case, it is clear thatefficient energy transfer from phycobilins to Chl a depends uponthe integrity of the membrane and is lost upon rehydrating driedtissues.The application of osmotic dehydration to marine algae ap-

pears to be a promising way to probe the effects of tissuedehydration on physiological functioning of macrophytic algae.

Acknowledgments-The authors would like to thank Dr. W. Z. Cande, Depart-mentof Botany, University ofCalifornia, Berkeley, forproviding a Zeiss microscopeequipped with Nomarski interference optics. C. M. S. was supported in part by a

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EFFECTS OF DRYING ON TWO SPECIES OF PORPHYRA

Miller Research Fellowship, in the Department of Botany, University ofCalifornia,Berkeley, and gratefully acknowledges that support.

LITERATURE CITED

1. BIEBL R 1952 Ecological and non-environmental constitutional resistance ofthe protoplasm of marine algae. J Mar Biol Assoc UK 31: 307-315

2. BIEBL R 1970 Vergleichende Untersuchungen zur Temperatur-resistenz vonMeeresalgen entlang der pazifischen Kuste Nordamerikas. Protoplasma 67:451-472

3. BRINKHuis B, N TEMPEL 1976 Photosynthesis and respiration of exposed salt-marsh fucoids. Mar Biol 34: 349-359

4. DRING MJ, FA BROWN 1982 Photosynthesis of intertidal brown algae duringand after periods of emersion: a renewed search for physiological causes ofzonation. Mar Biol Prog Ser 8: 301-308

5. FORK DC, G OQUIST 1981 The effects of desiccation on excitation energytransfer at physiological temperatures between the two photosystems of thered alga Porphvra perforata. Z Pflanzenphysiol 104: 385-393

6. FORK DC, GA FORD, B CATANZARO 1979 Measurements with a microproces-sor-based fluorescence spectrophotometer made on the blue-green alga An-acystis nidulans above and below the phase transition temperature. CarnegieInst Wash Year Book 78: 196-199

7. GANTT E 1981 Phycobilisomes. Annu Rev Plant Physiol 32: 327-3478. KREMER BP 1979 Photoassimilation products and osmoregulation in marine

Rhodophyceae. Z Pflanzenphysiol 93: 139-1479. MOHANTY P, DC FORK, JJ BRAND 1983 Calcium depletion affects energy

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transfer and prevents state changes in intact Anacystis. Photosyn Res 6: 349-361

10. MUNNS R, H GREENWAY, GO KIRST 1983 Halotolerant eukaryotes. In OLLange, PS Nobel, CB Osmond, H Zeigler, eds, Encyclopedia of PlantPhysiology, Vol 12. C New Series Physiological Plant Ecology III Responseto the Chemical and Biological Environment. Springer-Verlag, Berlin, pp60-135

1 1. OQuIST G, DC FORK 1982 Effects of desiccation on the excitation energydistribution from phycoerythrin to the two photosystems in the red algaPorphyra perforata. Physiol Plant 56: 56-62

12. REED RH, JC COLLINs, G RUSSEL 1980 The effects of salinity upon galactosyl-glycerol content and concentration of the marine red alga Porphyrapurpurea(Roth) C. Ag. J Exp Bot 31: 1539-1554

13. SANTARIUs K 1973 The protective effect of sugars on chloroplast membranesduring temperature and water stress and its relationship to frost, desiccationand heat resistance. Planta 113: 105-114

14. SATOH K, CM SMITH, DC FORK 1983 Effects of salinity on primary processesof photosynthesis in the red alga Porphyra perforata. Plant Physiol 73: 643-647

15. SMITH CM 1983 Photosynthetic tolerances to intertidal temperature and os-motic stress by select intertidal marine algae. PhD dissertation. StanfordUniversity, CA

16. SMITH CM 1984 Drying enhances tolerance to high temperature stress for anintertidal alga, Porphyra perforata. J. Ag. Am Zool 24: Suppl 29A, No. 137

17. WIENCKE C, A LAUCHLI 1980 Growth, cell volume and fine structure ofPorphyra umbilicalis in relation to osmotic tolerance. Planta 150: 303-31 1

18. WILTENS J, U SCHREIBER, W VIDAVER 1978 Chlorophyll fluorescence induc-tion: an indicator of photosynthetic activity in marine algae undergoingdesiccation. Can J Bot 56: 2782-2794

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