ALTERED ZYGOSPORE WALL ULTRASTRUCTURE CORRELATES WITH REDUCEDABIOTIC STRESS RESISTANCE IN A MUTANT STRAIN OF CHLAMYDOMONAS MONOICA

(CHLOROPHYTA)1

Patricia Daniel, Jessica Henley and Karen VanWinkle-Swift2

Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona 86011-5640, USA

The zygospore of Chlamydomonas is a diploidresting stage that provides protection from environ-mental extremes. The remarkable abiotic stressresistance of the zygospore can be explained, inpart, by the presence of a massive wall that includesa sporopollenin-containing surface layer (VanWinkle-Swift and Rickoll 1997). A Chlamydomonasmonoica Strehlow zygospore-specific mutant strain(D19) was obtained previously by screening for lossof chloroform resistance in zygospore populationsderived from self-mating of post-mutagenesisclones. Exposure of D19 zygospores to solar UVradiation or germicidal radiation also resulted in apronounced decrease in survival of D19 zygosporesrelative to wildtype zygospore survival. Similarly,resistance to NaCl-induced osmotic shock was re-duced in D19 zygospores, especially when exposedto very high (e.g., 20% w/v) salt concentrations. Ma-ture zygospores of C. monoica exhibit a UV-inducedblue surface autofluorescence that may indicate thepresence of phenolic wall components. The intensi-ty of zygospore autofluorescence was significantlyreduced in D19 zygospores. As revealed by TEM,the surface layer of mature homozygous D19 zy-gospores was disrupted, suggesting a defect in wallassembly. Zygospore-specific chloroform sensitivity,UV sensitivity, and reduced autofluorescence coseg-regated in tetrads derived from D19 heterozygotes(i.e., if a progeny clone from a cross involving D19and a normal strain was found to be chloroformsensitive, it was always also UV sensitive and showedreduced autofluorescence), indicating that all threecharacteristics were the consequence of the sameMendelian mutation.

Key index words: abiotic stress; cell wall; Chlamy-domonas monoica; sporopollenin; UV radiation;zygospore

We use the unicellular green alga Chlamydomonasmonoica as a model system for studying sporulation, astress response that provides resistance to a variety ofenvironmental extremes. All organisms must with-

stand transient environmental conditions that cannotsupport vegetative growth, and in unicellular algae,asexual cysts and sexual zygospores allow dormancy,and specialized walls help to protect these cells fromabiotic stress (Coleman 1983). The development ofprotective cell walls in the algae has been a key evolu-tionary step in the adaptation of green plants to ter-restrial habitats with more extreme environmentalfluctuations (Graham 1993, 1996).

The zygospore is the end product of sexual repro-duction in Chlamydomonas (Levine and Ebersold 1960,Harris 1989). Nitrogen-starved vegetative haploid cellsbecome gametic and fuse to produce a diploid zygote.Maturation of the diploid zygote into a zygospore oc-curs through a progression of cellular and physiologicalchanges, including the synthesis of an elaborately sculp-tured, multilayered wall (Triemer and Brown 1975,Cavalier-Smith 1976, VanWinkle-Swift and Rickoll1997). The zygospore wall, while providing protectionduring dormancy, remains responsive to the environ-mental signals that will later prompt spore germination.

Ultrastructural analysis by TEM reveals similaritiesbetween the C. monoica zygospore wall and the protec-tive walls of pollen grains, the spores of nonseedplants, and the walls of resistant cell types of diverseunicellular algae (VanWinkle-Swift and Rickoll 1997).Resistance to acetolysis, autofluorescence properties,spectral analyses, and morphological characteristics ofthe zygospore wall suggest the presence of a sporopol-lenin- or algaenan-like component (Kalina et al. 1993,VanWinkle-Swift and Rickoll 1997, Blokker et al.1999). The phenylpropanoid components of sporopol-lenin can account for the UV-absorbing properties(and related autofluorescence) of the zygospore wall,and have been implicated in the resistance of pollenand spores to solar radiation (Xiong et al. 1997,Rozema et al. 2001).

Although the surface layer of the C. monoica zygo-spore wall may be comprised of sporopollenin, thecomposition of the massive electron-opaque inner walllayer and its function(s) remain unknown. Ultrastruc-tural details of zygospore wall disintegration duringzygospore germination suggest the presence of uniquedomains within the wall layers that may be variouslyresistant to enzymatic digestion (Malmberg andVanWinkle-Swift 2001). Similarly, different layers ofthe zygospore wall may be essential for resistance tospecific abiotic stressors, a hypothesis that could be

1Received 17 July 2006. Accepted 8 November 2006.2Author for correspondence: e-mail Karen.VanWinkle-Swift

@nau.edu.

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J. Phycol. 43, 112–119 (2007)r 2007 by the Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2006.00313.x

tested by the isolation and characterization of mutantstrains with altered zygospore wall composition,assembly, or ultrastructure.

However, the zygospore is a diploid cell type, mak-ing the identification of zygospore-specific recessivemutations problematic in the most commonly studiedheterothallic species. We have chosen C. monoica as ourmodel because its homothallic mode of sexual repro-duction facilitates the isolation of zygospore mutants(VanWinkle-Swift and Burrascano 1983). Zygospore-specific mutations, even if recessive, are fully expressedfollowing homothallic self-mating within individualpost-mutagenesis clonal populations. Thus, haploidmutants of C. monoica will produce diploid zygosporesthat are homozygous for mutations of interest. Usingmutagenesis with UV light, or chemical mutagens, wehave obtained zygospore-specific mutants (zym, zygotematuration; or ger, germination) at frequencies as highas 10� 2. This high yield reflects the large number ofgene loci whose products are required for successfuldifferentiation of the diploid zygote into a mature zy-gospore, as well as the ease of selection in a homothallicspecies (VanWinkle-Swift et al. 1998).

Our earlier work has focused on the isolation of alarge collection of zym mutants and characterization ofthe collection by light microscopy (LM), complementa-tion testing, and tetrad analysis (VanWinkle-Swift andBurrascano 1983, VanWinkle-Swift et al. 1998). The zymmutants undergo normal gametogenesis, mating, andcell fusion. However, zygotes fail to mature fully, and vi-able zygospores are not produced. A second group ofmutants, collectively referred to as ger, show near-normalzygospore morphology at the LM level, but are defectivefor spore germination (Flynn 1985, VanWinkle-Swiftet al. 1988). We are now initiating more detailed ultra-structural studies on these mutant strains to identify andcharacterize those carrying mutations that directly relateto the enhanced zygospore stress resistance.

In the present study, we have focused on a mutantstrain, D19, that was originally described as a condi-tional germination mutant (Flynn 1985, VanWinkle-Swift et al. 1988). Although heavily walled zygosporeswere recovered from self-matings of the D19 strain,they failed to germinate when exposed to chloroform,a procedure routinely used to eliminate contaminatingunmated cells. However, when D19 zygospores werephysically separated from the background of haploidcells in the absence of chloroform selection, germin-ation occurred and viable progeny could be recovered.

The goal of the present study was to determine, first,whether the D19 strain was sensitive to other morenatural forms of environmental stress, and second,whether a more-detailed characterization of mutant zy-gospores would reveal abnormalities in zygospore wallultrastructure or composition that could account fortheir reduced environmental resistance.

MATERIALS AND METHODS

Strains and culture conditions. The wildtype strain (22B)of C. monoica used in this study is a high-mating-efficiency

derivative of the wildtype strain of C. monoica received fromthe Culture Collection of Algae and Protozoa (CCAP; Argyll,Scotland). The conditional germination mutant, D19, wasobtained by fluorodeoxyuridine mutagenesis of wildtypecells (Flynn 1985). The sta-1 strain was used in crosses toprovide an easily scored marker for distinguishing betweenhomozygous and heterozygous zygospores (see Rickollet al. 1998). Vegetative cultures of all strains were main-tained on agar-solidified HS medium (Harris 1989) undercontinuous cool-white fluorescent illumination (80 mmol pho-tons �m� 2 � s�1) at 201C. Gametogenesis was induced by sus-pension of vegetative cells in LPN medium as described byVanWinkle-Swift and Burrascano (1983). Mating cultureswere maintained at 201C under continuous illumination for7 days to allow time for completion of zygote maturation.

Purification of zygospores. Unmated cells and zygosporesfrom 7-day-old self-mated cultures were concentrated bycentrifugation at 3000g for 5 min. One milliliter of concen-trated cells was layered onto the surface of a 2 mL cushion of40% sucrose in a 15 mL sterile conical centrifuge tube. Aftercentrifugation at 3000g for 5 min, unmated cells formed apellet at the bottom of the tube. Zygospores remained at thesucrose/LPN interface and were removed by a sterile pipetteand washed two times in nitrogen-free BM (NBM) medium(Bischoff and Bold 1963). Purified and washed zygosporeswere used immediately, or the final pellet was covered with asmall volume of sterile NBM and stored at 41C until furthertesting.

Chloroform sensitivity. Purified zygospores in NBM wereplated as 20 mL aliquots on BM medium solidified with6 g �L� 1 agar plus 6 g �L� 1 gellan gum (BM-A/G). Once allfluid had been absorbed, the plates were inverted over a glasspetri dish containing ~10 mL of chloroform, exposing thezygospores to the vapors for 5–60 s. The plates were thenplaced under continuous illumination at 201C to inducegermination. Percent germination was determined by com-paring the number of colonies recovered after each chloro-form exposure with the total number of zygospores plated.The survival of vegetative cells was determined by removingcells from agar-solidified HS medium, suspending anddiluting the cells in NBM, and plating serial dilutions ontoBM-A/G. The survival of vegetative cells was determinedby comparing colony counts from control versus treatedsamples.

UV-radiation sensitivity. Serial dilutions of purified zy-gospores were plated as 20 mL aliquots onto BM-A/G, andthe plates were stored in darkness for 4 days at room tem-perature. The plates were then removed from the dark andirradiated with UV-C (254 nm; 8 W Sylvania shortwave UVbulb, UVP, Upland, CA, USA), or UV-B (315 nm; 15 W bulb,UVP) from a distance of 15 cm above the agar surface(380 W � cm�2). The time of exposure to radiation was var-ied from 10 to 300 s. The irradiated zygospores were thenreturned to dark storage for 18–24 h to prevent photoreac-tivation and were then placed under continuous illuminationat 201C to induce germination. The survival of vegetativecells was determined by removing cells from agar-solidifiedHS medium, suspending and diluting the cells in NBM, andplating serial dilutions onto BM-A/G. The plated cells werethen exposed to UV-B or UV-C radiation as above, stored indarkness for 18–24 h, and then returned to continuous light.Colony counts for irradiated versus unirradiated controlsfrom the same dilution were compared to determine percentsurvival.

Salt sensitivity. Purified zygospores were suspended for 1 hin aqueous NaCl solutions ranging in concentration from0% to 30% (w/v). Zygospores were removed from the salinesolutions by spin filtration at 12,000g for 15 s using 0.45 mmcellulose acetate Costar Spin-X filters (Sigma-Aldrich, St.

C. MONOICA ZYGOSPORE MUTANT 113

Louis, MO, USA). Treated zygospores were washed in NBMmedium, collected by spin filtration, and resuspended inNBM medium. Serial dilutions of the zygospore sampleswere prepared in NBM, and 20 mL aliquots of each dilutionwere plated on BM-A/G. The plated zygospores from appro-priate dilutions were counted using a dissection microscope.The plates were then held in darkness at room temperaturefor 3 days and were subsequently placed under continuousillumination at 201C to induce germination. Colonies derivedfrom germinated zygospores were counted 4–5 days afterinduction, and the counts were compared with the originalzygospore counts to arrive at the percent germination.

Fluorescence microscopy. Purified mature zygospores ofwildtype and mutant strains suspended in NBM mediumwere placed on a microscope slide, covered with a cover glass,and examined using a Zeiss Axioplan Phase/Fluorescencemicroscope (Carl Zeiss AG, Oberkochen, Germany) usingUV excitation and emission filters of 365 and 397 nm,respectively. Images were captured on an 800 ASA colorfilm at �400 magnification.

Fluorometry. The concentration of purified zygospores inNBM medium was determined by hemacytometer count. Allsamples were diluted to 5.8 � 105 zygospores �mL�1. Fluor-escence in 1 mL samples (with a 1 mL NBM sample as theblank) was measured using a Sequoia-Turner Model 450digital fluorometer (Sequoia-Turner Corp., Mountain View,CA, USA) with NB360 excitation and NB405 emission filters.The gain was set at 200. Zygospores were purified from threeindependent matings of each strain, and three readings weretaken and averaged for each sample.

TEM. Mating cultures of wildtype 22B and mutant D19were sampled at 36, 40, 44, 48, and 52 h after transfer ofvegetative cells to LPN medium. Cells (unmated cells andmaturing zygospores) were prepared for TEM as describedby VanWinkle-Swift and Rickoll (1997), with the modificationthat cells were concentrated and immobilized in alginatebeads following the initial fixation, as described by Fuentesand VanWinkle-Swift (2003).

Tetrad analysis. Tetrad analysis of a cross between the D19strain and a strain (sta-1) that was normal with regard to zy-gospore morphogenesis (D19þ ) was performed. The sta-1marker allowed us to distinguish between tetrads derivedfrom selfing within either parent strain and those derivedfrom crossing. Vegetative cells of the D19 mutant strain weretransferred to LPN medium along with cells from the sta-1(D19þ ) strain. After gametogenesis, mating, and zygote mat-uration, 20–30 mL aliquots of the mated population werestreaked on the surface of BM-A/G plates. The plates wereplaced in darkness for 3–7 days and were then inverted overa dish of chloroform for 20 s. Individual zygospores werepulled away from the background of unmated cells andallowed to germinate under continuous illumination. The

individual tetrad products released by germinated zy-gospores were separated using a fine glass needle and wereallowed to form colonies. Tetrad product clones were replica-plated to nutrient-free agar plates, incubated under contin-uous light for 4 days, and then inverted over iodine vapors(Rickoll et al. 1998) to identify tetrads derived from hetero-zygotes (D19/D19þ sta-1/sta-1þ ). Tetrad products from het-erozygotes were then self-mated, and the resultantzygospores were scored for chloroform sensitivity, UV sensi-tivity, and UV-induced autofluorescence.

RESULTS

Abiotic stress sensitivity of D19 mutant zygospores. Ex-posure to chloroform vapors is a technique routinelyused to select sexual zygospores from the back-ground of unmated Chlamydomonas gametes and veg-etative cells (Harris 1989). In a screening of clonesrecovered after fluorodeoxyuridine mutagenesis, theD19 strain was observed to produce morphologicallynormal zygospores at the level of LM, but no viableprogeny after exposure of the mated populations tochloroform vapors (Flynn 1985). When zygosporeswere individually manipulated away from the back-ground of unmated cells without the use of chloro-form selection, zygospore germination occurred andviable offspring were recovered.

The viability of wildtype vegetative cells of C. mono-ica was dramatically reduced by even a brief (e.g., 5 s)exposure to chloroform vapors, and vegetative cells ofthe D19 strain were similarly sensitive (Table 1). How-ever, wildtype zygospores showed little reduction ingermination efficiency after exposure to chloroformvapors for as long as 60 s. In contrast, zygospores fromthe D19 mutant strain were more sensitive to kill bychloroform vapors and did not survive a 20 s exposure(Table 1).

Although Chlamydomonas zygospores are unlikely tobe exposed to chloroform vapors in natural environ-ments, other forms of abiotic stress are common to themany environments where the alga occurs. A commonstress experienced by vegetative cells and zygospores isexposure to UV-B. As shown in Fig. 1, vegetative cellsof the D19 mutant and wildtype 22B strains showedsimilar sensitivities to UV-B or UV-C radiation. In con-trast, D19 mutant zygospores were more UV sensitive

TABLE 1. Chloroform sensitivity of vegetative cells and zygospores of Chlamydomonas monoica wild-type and mutant strains.

% Survivala

Chloroform exposure time (s)

0 5 10 20 30 60

Vegetative cells22B (wild-type) 100 25.6 � 11.0 1.2 � 1.1 0 0 0D19–18B 100 9.9 � 6.8 1.1 � 1.0 0 0 0

Zygospores22B (wild-type) 92.5 � 0.7 97.5 � 0.7 95.5 � 2.2 95.0 � 1.4 94.0 � 0 83.5 � 17.0D19–18B 95.0 � 1.4 30.0 � 12.0 4.1 � 4.1 0 0 0

aSurvival is measured as the efficiency of mitotic colony formation by vegetative cells or the percent meiotic germination of zy-gospores. Values are the means � the SE from three independent trials.

PATRICIA DANIEL ET AL.114

than wildtype zygospores. Again, the mutant pheno-type was zygospore-specific.

In nature, Chlamydomonas vegetative cells and zy-gospores may be exposed to microenvironments span-ning a wide range of salinities and water content. Notsurprisingly, wildtype C. monoica zygospores survivedexposure to hypersaline conditions that would be ex-pected to cause osmotic shock and cellular dehydration(Fig. 2). Suspension of vegetative cells of C. monoicain 5% NaCl for 1 h resulted in complete loss of viabilityin both wild-type and D19 strains (data not shown).A comparable treatment of zygospores had no signif-icant effect on zygospore germination upon returnto standard medium. The D19 mutant zygosporeswere also resistant to salt-induced osmotic shock atlower NaCl concentrations, but showed increasedsensitivity at higher concentrations (Fig. 2). The D19zygospores showed no survival after a 1 h exposureto 30% NaCl.

Characterization of the zygosopore wall of D19mutant zygospores. Fluorescence microscopy of ma-ture wildtype zygospores reveals a pronounced blueautofluorescence. We have proposed that this auto-fluorescence is associated with the zygospore walland, in particular, with the surface layer—asporopollenin-containing trilamellar sheath (Van-Winkle-Swift and Rickoll 1997, Malmberg andVanWinkle-Swift 2001). Zygospore-wall autofluores-

cence was dramatically reduced in the D19 strain ascompared with wildtype zygospores when viewed byfluorescence light microscopy (Fig. 3A). Relative zygo-spore fluorescence, as measured by fluorometry, wassignificantly reduced (P<0.0001; two-tailed t-test) inD19 zygospores as compared with wildtype zygosporefluorescence (Fig. 3B).

FIG. 1. Survival of wild-type and mutant vegetative cells and zygospores following exposure to UV radiation. Vegetative cells (rightpanels) or purified zygospores (left panels) derived from the wildtype 22B (&) and mutant D19 (}) strains were exposed to UV-B (top) orUV-C (bottom) radiation. Survival values are based on subsequent colony formation by vegetative cells and colony formation followingmeiotic germination of zygospores. Data are means � the SE from three-independent trials.

100

10

1

0.1

0.010 10 20 30 40

% NaCl

% S

urv

ival

FIG. 2. Relative sensitivity of wildtype and mutant zygosporesto NaCl-induced osmotic shock. Purified zygospores derivedfrom the wildtype 22B (&) and mutant D19 (}) strains were ex-posed to a range of concentrations of NaCl (w/v) in aqueous so-lutions for 1 h. Following exposure, survival was determined bymeasuring germination after transfer to standard medium. Dataare the means � the SE from three independent trials.

C. MONOICA ZYGOSPORE MUTANT 115

The reduced resistance to environmental stress andthe altered surface autofluorescence of D19 zygosporessuggested a wall defect. The ultrastructural stages insecondary zygospore wall assembly in wildtype zy-gospores have been described in detail previously(VanWinkle-Swift and Rickoll 1997). Abnormal wallassembly in D19 mutant zygospores was confirmed byTEM (Fig. 4).

Mature wildtype zygospores had a highly sculp-tured, reticulate surface, while the surface of D19 mu-tant zygospores was relatively smooth (Fig. 4, A and B).Early during maturation, a protective, but transient,primary zygote wall was synthesized beneath the ori-ginal gamete walls in both wildtype and mutant zy-gospores (Fig. 4, C and D; VanWinkle-Swift andRickoll 1997). The outer layer of the secondary zygo-spore wall was then assembled beneath the primaryzygote wall and included an electron-opaque ‘‘fuzzycoat’’ and an underlying electron translucent band(Fig. 4C). The outer sheath of D19 zygospores was dis-continuous and appeared as loosely associated seg-ments (Fig. 4D). Following assembly of the outersheath, electron-opaque material was secreted and ac-cumulated unevenly above the plasma membrane inwildtype zygospores, producing the peaks and valleyscharacteristic of the mature zygospore wall (Fig. 4, Eand G). In D19 zygospores, the inner wall material wasdistributed more evenly (Fig. 4F), and although a thickelectron-opaque inner wall layer was assembled, therewas little variation in thickness, and surface reticulationwas thus reduced (Fig. 4H). The surface sheath wasclearly aberrant with breaks in the fuzzy coat andirregular deposition of electron-translucent material.

Cosegregation of phenotypic traits. We performed tet-rad analysis of a cross between the D19 strain and astrain (sta-1) that was normal with regard to zygo-

spore morphogenesis (D19þ ). The sta-1 markerallowed us to distinguish between tetrads derivedfrom selfing within either parent strain and thosederived from crossing (see Materials and Methods).In 50 tetrads derived from crossing between the D19and D19þ strains, the chloroform-sensitive:resistantzygospore phenotype segregated 2:2, indicating thatthe phenotype was a consequence of a single nucleargene mutation. Furthermore, the chloroform sensi-tivity cosegregated with UV sensitivity and with re-duced zygospore wall autofluorescence. Thus, allthree aspects of the D19 phenotype (salt sensitivitywas not tested) were the consequence of the samemutation.

DISCUSSION

The zygospore of Chlamydomonas, protected by aunique, multilayered secondary wall, shows an en-hanced resistance to abiotic stress. To identify genesinvolved in zygospore wall synthesis and assembly, andto evaluate the role of wall components in conferringresistance to specific stressors, we have used the ho-mothallic species C. monoica to isolate zygospore-specific mutations that affect spore morphologyand viability (VanWinkle-Swift and Burrascano 1983,VanWinkle-Swift et al. 1998).

The D19 mutant was originally classified as a ger-mination (ger) mutant on the basis of near-normalmorphology at the level of LM and the conditional(chloroform sensitive) germination phenotype associ-ated with this strain (Flynn 1985, VanWinkle-Swiftet al. 1988). Using TEM of mature D19 zygospores,we have now shown that the surface layer of the sec-ondary wall is discontinuous. Although the assembly ofsegments of the trilamellar sheath, as seen in develop-

FIG. 3. UV-induced zygospore wall autofluorescence in wildtype and mutant C. monoica zygospores. (A) Phase contrast (a, c) and UV-fluorescence (b, d) images of wildtype 22B (a, b) and D19 mutant (c, d) zygospores; red autofluorescence is from chlorophyll; blueautofluorescence is associated with the zygospore wall. (B) Relative UV-induced fluorescence in wildtype and mutant zygospores asquantified by fluorometry. Data are the means � the SE from four independently prepared samples of purified zygospores from eachstrain.

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ing wildtype zygospores (VanWinkle-Swift and Rickoll1997), appears to occur normally early during zygotematuration in the D19 strain, later steps in assembly

and/or maintenance of the sheath are abnormal. Thus,the germination defect in D19 zygospores is a second-ary consequence of abnormal wall development, and

FIG. 4. TEM of wall development during the maturation of wildtype and mutant zygotes. (A, B) Mature zygospores derived from thewildtype 22B (A) and mutant D19 (B) strains. Scale bars, 1mm. (C, D) Early zygote maturation includes primary wall (short arrow) andtrilamellar sheath (long arrow) synthesis and assembly in wildtype 22B (C) and mutant D19 (D) zygotes. Scale bars, 200 nm. (E, F)Deposition of the inner secondary wall layer (arrow) occurs in both wildtype 22B (E) and D19 (F) maturing zygotes. Scale bars, 200 nm.(G, H) Detail of the mature secondary zygospore wall showing the peaks and valleys typical of wildtype (G) zygospores, and thedisrupted surface layer (trilamellar sheath) in mutant D19 (H) zygospores. Scale bars, 100 nm.

C. MONOICA ZYGOSPORE MUTANT 117

the mutant is more aptly described as a zygote matur-ation (zym) mutant.

Within 6 h after induction of germination, C. monoicawildtype zygospores become sensitive to chloroform(Malmberg and VanWinkle-Swift 2001). The timing ofacquired chloroform sensitivity correlates with the ap-pearance of isolated lesions in the surface layer (trila-mellar sheath) of the zygospore wall during the earlystages of germination (Malmberg and VanWinkle-Swift2001), before breakdown of the inner wall layer. Inlight of this observation, which suggests that chloro-form vapors can permeate the inner wall layer but notthe outer sheath, it is not surprising that the disruptedsurface layer in D19 mutant zygospores is associatedwith a chloroform-sensitive phenotype.

In wildtype zygospores, the secretion of inner wallmaterial occurs at discrete sites along the plasma mem-brane soon after the formation of an intact outersheath (VanWinkle-Swift and Rickoll 1997). Continuedsecretion and assembly of inner wall precursors isassociated with the formation of distinct peaks andvalleys and is responsible for the surface reticulationcharacteristic of the mature wildtype zygospore. InD19 zygospores, the deposition of inner wall materialoccurs more uniformly around the cell periphery. Thisdeposition may be a secondary consequence of disin-tegration or aberrant assembly of the outer wall layer ifthe D19 gene functions in outer sheath assembly. How-ever, we cannot rule out the possibility that the D19gene encodes a function that is essential for establish-ing a pattern of deposition of inner wall componentsthat stabilizes the outer wall layer.

The discontinuity of the surface sheath may explainthe reduced UV-induced autofluorescence of D19 mu-tant zygospores and their increased sensitivity to UVradiation. Similarly, increased sensitivity of D19 zy-gospores to salt stress could be a consequence of in-creased permeability of the outer wall layer, increasedexposure of the inner wall layer to osmotic shock, orleaching of protective components from the inner walllayer(s).

The zygospore wall of C. monoica is resistant to ac-etolysis (VanWinkle-Swift and Rickoll 1997), a com-mon diagnostic test for sporopollenin (Shaw 1971),and the resistant residue is autofluorescent when ex-cited by UV light (VanWinkle-Swift and Rickoll 1997).The blue autofluorescence of wildtype untreated (via-ble) zygospores, which is weaker in D19 zygospores,may be derived from sporopollenin or from other UV-absorbing phenolic compounds loosely associated withor covalently linked to structural wall polymers.

In higher plants, UV-absorbing cytoplasmic flavon-oids and sinapate esters associated with the cell wallhelp to protect vegetative tissue from the damagingeffects of solar UV radiation (Landry et al. 1995,Jansen et al. 1998). The sporopollenin of pollen ex-ines and phenolic components in the overlying pollencoat (tryphine) may also act as sunscreens (Day andDemchik 1996, Piffanelli et al. 1998, Rozema et al.2001). Not surprisingly, phenolic compounds increase

in abundance in many plant tissues in response to UV-B irradiation (Day and Vogelmann 1995, Landry et al.1995, Jansen et al. 1998, Santos et al. 1998, Rozemaet al. 2002).

In microscopic algae, the presence of sporopollenin-containing walls and/or the radiation-induced synthe-sis of mycosporine-like amino acids (MAAs) serve toprotect cells from UV radiation (Xiong et al. 1997,1999, Sinha et al. 2001, Rozema et al. 2002). The ev-olution of strategies for limiting the damaging effectsof UV-B has been crucial to the successful establish-ment of plants in terrestrial environments (Graham1993, 1996, Rozema et al. 1997).

The C. monoica zygospore may serve as an algalmodel for studying the morphogenesis of an intricatelypatterned and chemically resistant cell wall, and forevaluating the role of wall components in conferringabiotic stress resistance. Many zygospore-specific mu-tants of C. monoica are available (VanWinkle-Swiftet al. 1988, VanWinkle-Swift et al. 1998), and ongoingultrastrucural analyses are helping to identify thosewith wall-specific abnormalities such as the D19 straindescribed here. These genetic and ultrastructural stud-ies, in conjunction with more-detailed biochemicalcharacterization of the zygospore wall, should provideinsights into the unique functions of individual walllayers and components. Conditional mutants, such asD19, in which zygospore viability varies with environ-mental conditions, are especially useful for identifyingthe cellular and molecular factors responsible for abi-otic stress resistance. Ultimately, the establishment oftransformation protocols for C. monoica (in progress)will allow the cloning of relevant genes by complemen-tation of mutant phenotypes—a critical step towardidentification of their cellular functions.

This work was supported by National Institutes of Healthgrant 1R15 GM071374-01 to K. V. W.-S., a predoctoral fel-lowship to P. D. from the ARCS Foundation, and an intra-mural Hooper Undergraduate Research Award to J. H. Wethank Marilee Sellers of the NAU Core Histology and ImagingFacility for technical advice and instrumentation support inthe area of TEM.

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