Response to the sexual pheromone and wounding in the green alga volvox: induction of an extracellular glycoprotein consisting almost exclusively of hydroxyproline.

The extracellular matrix (ECM) of Volvox is modified during development or in response to external stimuli, like the sex-inducing pheromone. It has recently been demonstrated that a number of genes triggered by the sex-inducing pheromone are also inducible by wounding. By differential screening of a cDNA library, a novel gene was identified that is transcribed in response to the pheromone. Its gene product was characterized as an ECM glycoprotein with a striking feature: it exhibits a hydroxyproline content of 68% and therefore is an extreme member of the family of hydroxyproline-rich glycoproteins (HRGPs). HRGPs are known as constituents of higher plant ECMs and seem to function as structural barriers in defense responses. The Volvox HRGP is also found to be inducible by wounding. This indicates that the wound response scenarios of higher plants and multicellular green algae may be evolutionary related.

The extracellular matrix (ECM) of Volvox is modified during development or in response to external stimuli, like the sex-inducing pheromone. It has recently been demonstrated that a number of genes triggered by the sex-inducing pheromone are also inducible by wounding. By differential screening of a cDNA library, a novel gene was identified that is transcribed in response to the pheromone. Its gene product was characterized as an ECM glycoprotein with a striking feature: it exhibits a hydroxyproline content of 68% and therefore is an extreme member of the family of hydroxyproline-rich glycoproteins (HRGPs). HRGPs are known as constituents of higher plant ECMs and seem to function as structural barriers in defense responses. The Volvox HRGP is also found to be inducible by wounding. This indicates that the wound response scenarios of higher plants and multicellular green algae may be evolutionary related.
The evolution of a complex extracellular matrix (ECM) 1 from a simple cell wall was one of the prerequisites to promote the transition from unicellularity to multicellularity. The volvocine algae provide the unique opportunity for exploring the pathways that led from a simple cell wall to a complex ECM that stabilizes the shape of an organism and mediates many developmental responses of cells to internal as well as external stimuli. The volvocine algae range in complexity from unicellular Chlamydomonas to multicellular organisms, with differentiated cells and complete division of labor, in the genus Volvox. The asexually growing organism of Volvox carteri is composed of only two cell types: 2000 -4000 biflagellate Chlamydomonas-like somatic cells are arranged in a monolayer at the surface of a hollow sphere (1, 2) and 16 much larger reproductive cells ("gonidia") lie just below the somatic cell sheet. Volvox cells are surrounded and held together by a glycoprotein-rich ECM (reviewed in Refs. 3 and 4). Cell walls and ECMs of the volvocine algae are assembled entirely from glycoproteins (5) and a high content of hydroxyproline has been detected. Hydroxyproline-rich glycoproteins (HRGPs) represent a constituent of higher plant ECMs, and much work has been done to analyze the structures of these proteins (6 -11). However, there are few examples in the literature where multiple ECM proteins have been examined in molecular detail from a single species or from closely related species. This approach has been initiated with volvocine algae to allow a more integrated approach to elucidate the structure, assembly, and function of ECM proteins.
A remarkably rapid remodelling of the ECM is observed under the influence of the sex-inducing pheromone (a glycoprotein) that triggers initiation of the sexual life cycle of Volvox carteri (12)(13)(14). In particular, synthesis of some members of the pherophorin family of ECM proteins (15)(16)(17) is strongly induced by the pheromone. Pherophorins are ECM glycoproteins that contain a C-terminal domain with homology to the sexinducing pheromone.
By differential screening of a cDNA library, additional genes were recently identified that are transcribed under the control of the sex-inducing pheromone (18). Unexpectedly, genes were found, in addition to those encoding the pherophorins, that encode extracellular chitinases and proteinases. In higher plants, similar protein families are known to play an important role in defense against fungi. Indeed, it could be demonstrated that the same set of genes triggered by the sex-inducing pheromone is also inducible by wounding of Volvox spheroids.
Pheromone-induced changes in the composition of the ECM have been characterized in detail within the cellular zone of the ECM (12,13,15,16,19) and to a lesser extent within the deep zone (DZ) (17), which contains all ECM components internal to the cellular zone (for nomenclature see Ref. 3). The DZ appears as a relatively amorphous component that fills the deepest regions of the spheroid and that may constitute more than 90% of the total volume of the organism. In this paper, we characterize a HRGP exhibiting an extreme composition that is expressed in response to the sex-inducing pheromone and to wounding and that is part of the DZ compartment of the ECM.  (21). Strain 153-48 was grown in the presence of 1 mM NH 4 Cl. The sex-inducing pheromone was used as described (22). Differential Screening of a cDNA Library-Total RNA was isolated from V. carteri spheroids (HK10) at various times after the addition of the sex-inducing pheromone. RNA samples isolated 3, 6, and 12 h after the application of the pheromone were pooled, and a cDNA library was prepared from the corresponding poly(A) ϩ mRNA preparation (18) by using the ZAP cDNA synthesis kit (Stratagene, La Jolla, CA). Replica filters were probed with 32 P-labeled cDNA prepared from polyadeny-* This work was supported by Grant SFB 521 from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AJ242540.
‡ These authors contributed equally to this work and are considered first authors.
Cloning of the dz-hrgp Gene-The dz-hrgp cDNA fragment obtained by differential screening of the cDNA library was used as a probe to screen a V. carteri genomic library (19) in EMBL 3 (24). Cloning of the dz-hrgp gene followed standard techniques (23).
PCR Amplification of dz-hrgp cDNA Fragments-RNA from sexually induced (2.5 h) V. carteri spheroids (HK10) was used to construct a cDNA library covalently linked to magnetic beads according to the instructions of the manufacturer of the beads (Deutsche Dynal, Hamburg, Germany), and cDNA fragments of dz-hrgp were amplified by PCR. Alternatively, cDNA fragments were amplified by reverse transcription PCR as described (25). RACE-PCR technique was performed as described (26).
DNA Sequencing-Genomic and cDNA clones of dz-hrgp were mapped with standard restriction enzymes, and restriction fragments were subcloned. To create targeted breakpoints for DNA sequencing, these subclones were digested unidirectionally with exonuclease III (27). This was done from both sides of the subclones. Products were transformed into ultracompetent Escherichia coli cells (Epicurian Coli, Stratagene). The precise length of a given insert was determined by gel electrophoresis. Sequencing from both directions was done by cycle sequencing (28,29) and isothermal sequencing (30) using vector-based oligonucleotide primers that were end labeled with [␥-32 P]ATP. Sequencing of GC-rich stretches was improved by the addition of Me 2 SO (31) and by using nucleotide analogues (32).
Northern Blot Analysis-Total RNA (10 g) from V. carteri strain HK10 was separated on a 1.0% denaturing gel (23), vacuum-blotted, UV-cross-linked onto Hybond-N membrane (Amersham Pharmacia Biotech), and hybridized with a 0.6-kb 32 P-labeled cDNA fragment of dzhrgp. This cDNA fragment came from the 3Ј-untranslated region of dz-hrgp to prevent cross-reactions with other polyproline-encoding genes.
Reverse Transcription-PCR-Volvox spheroids were incubated with or without the presence of the sex-inducing pheromone or were wounded by forcing a concentrated Volvox suspension through a 0.5-mm hypodermic needle. Reverse transcription-PCR from 20 Volvox spheroids was performed as described (25). The antisense oligonucleotide primer 5Ј-GTGTTTCCACCAGTGCGA and the sense oligonucleotide primer 5Ј-GAGCCATGTGGAAAGTCG were used for PCR amplification of a 117-base pair dz-hrgp cDNA fragment. Products of PCR amplification were cloned and sequenced.
Construction of Chimeric Genes-The fusion regions of the chimeric dz-hrgp promoter-dz-hrgp gene or the chimeric dz-hrgp-arylsulfatase gene were generated by the recombinant PCR technique (33). The final construction was performed by standard techniques (23).
Genomic PCR-Genomic PCR was used to confirm stable transformation of Volvox. 50 spheroids were selected under a stereomicroscope and transferred into 10 l of sterile lysis buffer (0.1 M NaOH, 2.0 M NaCl, 0.5% SDS). After 5 min at 95°C 200 l of 50 mM Tris/HCl, pH 7.5, were added immediately. 2 l of the resulting lysate was used for PCR (in a total volume of 100 l). PCR was performed by standard protocol. Products of PCR amplification were cloned into the SmaI site of pUC18 and sequenced.
Preparation of Anti-DZ-HRGP Antiserum-The peptide PRRSPV-VALVETC (amino acids 26 -37 of DZ-HRGP) with an artificial cysteine at the C-terminal end was synthesized by using Fmoc (9-fluorenylmethyloxycarbonyl) amino acid derivatives. The peptide was purified on a reversed phase C 18 HPLC column (Nucleosil 100 -7, 7 m; Macherey-Nagel, Dü ren, Germany). The predicted molecular mass and the sequence of the peptide were confirmed by electrospray mass spectrometry and by Edman degradation. The synthetic peptide was covalently linked to a maleimide-activated carrier protein (keyhole limpet hemocyanin) via the SH group of the artificial cysteine and used to raise polyclonal antibodies in rabbit. Antibodies were purified by protein G-Sepharose column chromatography (Amersham Pharmacia Biotech). Further purification of anti DZ-HRGP antiserum was on an affinity column (Sulfolink Coupling Gel, Pierce) with covalently linked DZ-HRGP peptide. The column was produced and handled as described (36). The antibodies were tested by Western blot analysis, using the synthetic peptide (this time covalently linked to bovine serum albumin) as an antigen.
Localization of DZ-HRGP-Whole Volvox spheroids were separated into defined fractions to allow localization of DZ-HRGP. A DZ extract was prepared as described below. The remaining material (intact cells and cell-bound ECM) was extracted with 2 M NaCl (2 h) and then disrupted ultrasonically (Sonifier B15, Branson, Danbury, CT). Soluble and insoluble components were separated by ultracentrifugation (100,000 ϫ g, 30 min). All fractions were lyophilized, deglycosylated by anhydrous HF (37), and analyzed by a Western blot using the polyclonal DZ-HRGP antibody.
Purification of DZ-HRGP from Volvox-Sexually induced Volvox spheroids from three 20 l cultures were harvested at the stage of embryogenesis by filtration on a 100-m mesh nylon screen. The spheroids were broken up by forcing them through a 0.5-mm hypodermic needle. The disrupted spheroids were centrifuged at 25,000 ϫ g for 30 min. The supernatant (DZ extract) was brought to 50 mM Tris/HCl, pH 9.0, 10 mM NaCl and applied to a QAE-Sephadex A-25 anion exchange column (Amersham Pharmacia Biotech) equilibrated with the same buffer. Elution was performed with 250, 500 and 800 mM NaCl. Fractions containing DZ-HRGP antigen were dialyzed against 4 mM NH 4 HCO 3 and lyophilized. The dried material was deglycosylated by anhydrous HF (37), applied to SDS-PAGE, and blotted onto polyvinylidene difluoride membrane. The membrane was stained with Coomassie Blue R-250 (Serva, Heidelberg, Germany) in 50% methanol, destained with 50% methanol/10% acetic acid, and washed with water. The band corresponding to the DZ-HRGP antigen was cut out and sequenced using an automated gas-phase peptide sequencer (Applied Biosystems, Foster City, CA).
Amino Acid Analysis and Mass Spectrometry-Amino acid analysis was performed as described by Cohen and Strydom (39). Molecular masses of fractions of interest were determined by electrospray mass spectrometry using a SSQ 7000 mass spectrometer (Finnigan).

RESULTS
Differential Screening-Total RNA was isolated from V. carteri spheroids (female HK10) harvested at various times after the addition of the sex-inducing pheromone. RNA samples isolated 3, 6, and 12 h after the application of the pheromone were pooled, and a cDNA library (ZAP) was constructed from the corresponding poly(A) ϩ mRNA preparation. Repeated differential screenings of the cDNA library with cDNA derived from asexual versus sexually induced organisms resulted in the detection of novel clones in addition to the already known members of the pherophorin family (15)(16)(17) and to the chitinase and the cysteine protease described recently (18). One of these novel clones was named dz-hrgp for reasons explained below. The kinetics of dz-hrgp mRNA accumulation in response to the sex-inducing pheromone was analyzed by Northern blotting. As shown in Fig. 1, hybridizing RNA started to accumulate only about 30 min after pheromone treatment and reached its maximum 2 h later. No significant signals were observed in asexually growing organisms. The mRNA detected by the dz-hrgp cDNA fragment is ϳ2.0 kb in length.
DNA Sequencing and Deduced Amino Acid Sequence-To extend the cDNA sequence information obtained from the originally isolated 0.6-kb cDNA, the RACE-PCR (26) was used to obtain the missing 5Ј stretches. But the RACE-PCR yielded only a 0.25-kb fragment because a stretch of unusually high (G)C-content caused premature termination of reverse transcription. To circumvent this problem, the dz-hrgp cDNA fragment was used to clone the corresponding genomic DNA. Sequencing of this unusual stretch of genomic DNA also produced particular problems and was only possible by the application of special techniques like creation of targeted breakpoints for DNA sequencing by exonuclease III digestion of subclones (for details, see "Experimental Procedures"). The strategy applied to collect the complete nucleotide sequence of dz-hrgp cDNA is shown in Fig. 2a. The sequence was submitted to the Gen-Bank TM /EBI Data Bank with accession number AJ242540. The dz-hrgp gene contains a single intron within its coding region. The deduced amino acid sequence for the DZ-HRGP is shown in Fig. 2b. This amino acid sequence exhibits striking features. The open reading frame encodes a polypeptide 409 amino acid residues in length, including a typical signal sequence. (Hydroxy-)proline constitutes 68% of the amino acid residues of the mature polypeptide. Several stretches of up to 14 (hydroxy)proline residues and numerous repeats of Ser-(Pro) 3 as well as (Ser/Arg)-(Pro) 4 elements are special features of this gene product. These data (and the data presented below) indicate a close relation of this novel Volvox protein to a well known protein family, namely, to the HRGPs of higher plants (6 -11). The reason for giving DZ-HRGP its name was this relationship and its localization within the DZ of the Volvox ECM (see below).
Because of the extended proline stretches of DZ-HRGP there are only short amino acid sequences near both the N and C termini of the polypeptide, which could serve as a suitable antigen in DZ-HRGP antibody production to prevent cross-reactions with other proline-rich ECM glycoproteins. Therefore, a sequence derived from the N-terminal end of DZ-HRGP was used to synthesize the peptide PRRSPVVALVETC. An artificial cysteine at the C terminus of this peptide simplified coupling to a carrier protein. Peptide specific polyclonal antibodies were raised in rabbit.
Identification and Homologous Overexpression of DZ-HRGP-The deduced DZ-HRGP amino acid sequence includes a typical signal peptide indicating an extracellular localization of DZ-HRGP. The peptide-specific antibody was used to search for DZ-HRGP in different extracts prepared from sexually induced Volvox spheroids. However, neither complete lysates nor ECM fractions produced any signal in immunodetection experiments. The putative extracellular localization of DZ-HRGP suggests extensive glycosylation of hydroxyproline residues, and this in turn could prevent immunodetection by our peptidespecific antibody. Therefore, the components of Volvox extracts were deglycosylated by treatment with anhydrous HF. Indeed, after deglycosylation, positive signals at ϳ150 kDa could be obtained in extracts from complete Volvox spheroids as well as in an ECM extract representing the DZ of Volvox ECM. The material of the DZ is selectively released by mild mechanical stress as may be exerted by forcing Volvox spheroids through a hypodermic needle.
Because Western blots yielded only a weak DZ-HRGP signal, overexpression of DZ-HRGP in Volvox was thought to get sufficient amounts of DZ-HRGP for structural studies. Random integration by illegitimate recombination events is the preferred mode of DNA integration into the Volvox genome, and transformants often integrate multiple copies of the plasmids used for transformation (34). Therefore, transgenic Volvox were generated that express additional copies of the dz-hrgp gene under the control of its own promoter. Stable transformants were produced as described previously (25,34,35,40). The transgenic Volvox strain did not show any visible change in phenotype, but the expression rate of DZ-HRGP was clearly higher than in wild-type algae. Again, the polyclonal DZ-HRGP antibody leads to a positive immunosignal at ϳ150 kDa after deglycosylation of extracts from sexually induced transformants. ϳ50% of DZ-HRGP is liberated just by forcing the Volvox spheroids through a hypodermic needle (DZ extract); all of the remaining DZ-HRGP is extracted in the presence of 2 M NaCl.
A molecular mass of 39.6 kDa is calculated for the mature polypeptide chain of DZ-HRGP (Fig. 2b), but this is much less than the apparent molecular mass of deglycosylated DZ-HRGP (ϳ150 kDa) shown on Western blot gels (Fig. 3b). The extreme (hydroxy-)proline content of DZ-HRGP explains the difference between the observed and calculated molecular masses, because stretches of poly-(hydroxy-)proline have a reduced ability to bind SDS (41).
Purification of DZ-HRGP-To prove the identity of the immunoreactive material and the DZ-HRGP, the components of the DZ extract of the ECM were fractionated by ion exchange chromatography (QAE-Sephadex). Because proteins from the DZ are hardly stained with standard procedures on SDS-PAGE gels, the Volvox spheroids were grown in the presence of [ 14 C]bicarbonate prior to preparation of the DZ extract, and the SDS-PAGE gels were analyzed by fluorography. As demonstrated by analytical SDS-PAGE, the QAE-Sephadex chromatography separated two main protein species of the deep zone extract completely from each other. These protein species exhibit apparent molecular masses of ϳ300 and ϳ240 kDa and elute at 500 and at 800 mM NaCl, respectively (Fig. 3a). The material of both fractions was then deglycosylated by treatment with anhydrous HF and fractionated by 6% SDS-PAGE. After blotting, an immunoreactive polypeptide with an apparent molecular mass of ϳ150 kDa could be detected in the 500 mM NaCl fraction (Fig. 3b). To confirm that it is indeed the ϳ300-kDa protein (glycosylated) that produces the ϳ150-kDa immunosignal (deglycosylated), the ϳ300-kDa protein was further purified by excision from a SDS-polyacrylamide gel. After elution the ϳ300-kDa protein was deglycosylated and subjected to SDS-PAGE. Again, the ϳ150-kDa immunosignal was detectable in a Western blot (data not shown). The immunoreactive polypeptide was subjected to automated Edman degradation, resulting in the N-terminal sequence Ala-Hyp-Ala-Arg-Lys-Hyp-Hyp-Hyp-Arg-Arg-Ser-Hyp, matching the N-terminal sequence deduced for mature DZ-HRGP. Remarkably, even the very first proline residue of the polypeptide turned out to be posttranslationally modified to hydroxyproline. Amino acid analyses of DZ-HRGP resulted in molar ratios of the predominant amino acids hydroxyproline, arginine, and serine of 10: 1:1.4. On the basis of the cDNA sequence the ratios of (hydroxy)-proline, arginine, and serine were calculated as 10.2: 1:1.6. All the other amino acids within DZ-HRGP could not be quantified exactly in amino acid analyses because of the small amounts detected. As shown in Fig. 4, all of the prolines of DZ-HRGP are found to be modified to hydroxyproline.
Carbohydrate Composition of DZ-HRGP-The carbohydrate composition of DZ-HRGP was determined by radio gas chromatography. DZ-HRGP purified from Volvox spheroids grown in the presence of [ 14 C]bicarbonate was hydrolyzed, and the resulting monosaccharides were analyzed as the alditol acetates. DZ-HRGP contains the neutral sugars arabinose and galactose in a 2:1 ratio (Fig. 5).
The Promoter of dz-hrgp Gene Mediates Pheromone-dependent Transcription-To examine the properties of the dz-hrgp promoter, the 5Ј-nontranslated region (ϳ3 kb) of dz-hrgp was placed in front of a reporter gene, the arylsulfatase gene from Volvox (25,42) (Fig. 6a). In wild-type Volvox, arylsulfatase is only expressed under sulfur starvation; no activity is detectable in organisms grown in sulfate-containing medium (42).
After transformation of Volvox with the chimeric dz-hrgp/ arylsulfatase gene, the reverse transcription-PCR technique was used to verify the existence of hybrid mRNA in transformants (see "Experimental Procedures"). Volvox transformants containing the arylsulfatase gene under the control of the dzhrgp promoter were incubated with or without the sex-inducing pheromone in the presence of the chromogenic enzyme sub-strate 4-nitrocatechol sulfate. Arylsulfatase activity was determined photometrically by measuring the absorbance of the liberated 4-nitrocatechol. After treatment with the sex-inducing pheromone, only transformants exhibited enzyme activity (Fig. 6b). Thus, the promoter fragment from the dz-hrgp gene is sufficient to mediate transcription in response to the sex-inducing pheromone.
Transcription of dz-hrgp Gene Is Cell Type-specific-In the Northern blotting experiments described above whole Volvox spheroids were used for RNA preparation. To investigate whether transcription of the dz-hrgp gene is cell type-specific, both cell types of (sexually induced) Volvox spheroids, somatic and reproductive, were separated from each other by size frac- tionation. RNA from both cell types was extracted and reverse transcribed, and a dz-hrgp cDNA fragment was amplified by PCR. Transcription of the dz-hrgp gene was detectable mainly in somatic cells, as shown in Fig. 7a.
Wounding Induces Transcription of dz-hrgp Gene-As demonstrated recently (18), all genes known so far to be under the control of the sex-inducing pheromone are also triggered by wounding. This observation led us to investigate whether transcription of dz-hrgp gene is also responsive to wounding. Volvox spheroids were slit by mild mechanical stress simply by forcing them through a hypodermic needle. 2.5 h later total RNA from 20 spheroids was extracted and reverse transcribed, and a dz-hrgp cDNA fragment amplified by PCR. As shown in Fig. 7b, transcription of dz-hrgp gene is not detectable in asexually growing Volvox colonies. In contrast, the sex-inducing pheromone as well as wounding induces transcription of this gene (Fig. 7b). Wounding even appears to stimulate a higher rate of transcription as compared with the induction by the sex-inducing pheromone. DISCUSSION This paper describes the structure and properties of a novel component of the Volvox ECM with the striking feature of being composed of 68% hydroxyproline. Some of these hydroxyprolines are arranged in Ser-(Pro) 3 and Ser-(Pro) 4 elements that are typical of higher plant extensins (9). Bradley et al. (43) noted that tissue wounding in higher plants selectively stimulates the expression of tyrosine-rich extensins. In this respect, the algal DZ-HRPG is different because it contains only a single tyrosine residue. Similarly, it does not perfectly fit into the general class of higher plant extensins because the predominant basic amino acid lysine is replaced by arginine in the algal polypeptide. To our knowledge, there is no other protein described with a (hydroxy)-proline content as high as 68%. For example, the higher plant extensins with the highest (hydroxy)-proline contents are those of Vigna unguiculata (44), Nicotiana tabacum (45), and Gossypium barbadense (46), exhibiting 53, 44, and 40%, respectively. In the animal kingdom the mini-collagen of Hydra attenuata (47) exhibits 44%.
A typical feature of algal ECM proteins characterized so far is a strictly modular composition, where hydroxyproline-rich (HR) sequences seem to serve as rod-shaped spacers separating modules that are completely devoid of hydroxyproline residues. It has been suggested that these hydroxyproline-rich stretches define a HR module family combining more specialized modules to yield chimeric and multifunctional ECM proteins (4). In this sense, DZ-HRGP represents the first volvocacean ECM glycoprotein being only composed of one (or more?) HR module(s). Electronmicroscopic studies have shown that main parts of the volvocacean ECM consist of a network of fibrous structures (48,49). Most probably, the rod-shaped HR modules mainly have a structural function and serve as building blocks to create these defined framework of the ECM. Where analyzed in more detail, these modules were found to be targets for extensive posttranslational modifications. Among the modifications identified in Volvox are O-glycosylations with oligoarabinosides, attachment of saccharides containing phosphodiester bridges between arabinose residues, and in a single case, the additional attachment of a highly sulfated arabinomannan (19). As analyzed in more detail for the ECM protein SSG 185, the HR module is also involved in covalent crosslinking of the monomeric units (19). If DZ-HRPG serves a similar function, this rod-shaped molecule could be involved in the creation of the fibrous networks observed in the deep zone compartment (3). The fact that overexpression of DZ-HRPG did not create an aberrant ECM morphology could indicate the participation of a second ECM molecule in the cross-linking reaction. In this case, only the concomitant overexpression of both ECM partners interacting in a stoichiometric relation would be expected to cause a visible phenotype.
The natural resistance of higher plants to diseases involves an array of inducible defense responses, including synthesis of extracellular hydrolytic enzymes such as proteases and chiti-FIG. 6. The promoter region of the dz-hrgp gene mediates transcription control by the sex-inducing pheromone. a, structure of the chimeric gene containing the dz-hrgp promoter region and the arylsulfatase reporter gene (genomic clone). b, photometric arylsulfatase activity assay (25) from transformants. Algae were disrupted by ultrasonic treatment, and the lysates were assayed using 4-nitrocatechol sulfate as a chromogenic substrate (optical density at 515 nm produced in 30 min by 50 algae/ml). Extracts from asexually grown (Ϫ) or sexually induced (ϩ) Volvox algae are shown. Transformants (T1 and T2) are compared with the nitA Ϫ strain 153-48 (N Ϫ ) used as DNA recipient and to wild-type HK10 algae (WT).

FIG. 7. Cellular localization and induction of dz-hrgp mRNA.
dz-hrgp mRNA was detected by reverse transcription and subsequent PCR amplification. A 117-base pair cDNA fragment of dz-hrgp was expected if the intron within this fragment was spliced correctly. a, somatic and reproductive cells of sexually induced Volvox spheroids were investigated separately to identify the cellular localization of dz-hrgp mRNA. b, vegetatively grown Volvox spheroids, spheroids after incubation with the sex-inducing pheromone (for 2.5 h), or vegetatively grown spheroids 2.5 h after wounding were investigated. nases and the accumulation of HRGPs within the ECM. The latter glycoproteins are hypothesized to function in defense as structural barriers (50) or as specific microbial agglutinins (51,52) against pathogen attack. Surprisingly, the simple multicellular green alga Volvox responds to wounding in much the same way as observed in higher plants. As was demonstrated recently (18), Volvox responds to wounding with the synthesis of a chitinase as well as a protease that is combined with chitin-binding modules. With the additional demonstration of a typical HRGP that is produced in response to wounding, it now appears that much of the response scenario found in higher plants already exists in multicellular green algae. Even more surprising is the fact that these algal pathways are also triggered by the sex-inducing pheromone. However, Kirk and Kirk (53) were able to demonstrate that synthesis of the sex-inducing pheromone can be triggered in somatic cells by a short heat shock applied to asexually growing organisms. This response induces the production of dormant zygotes that survive unfavorable conditions like drought. Although wounding is unable to induce pheromone production, similar biochemical responses are observed after wounding and pheromone application (18). As induction of sexuality and subsequent production of zygotes obviously is part of the strategy of the organism to escape from environmental stress, it appears to make sense that apparently completely different stimuli (wounding and pheromone treatment) cause up-regulation of the same set of genes. Further studies on this algal system should confirm or deny an evolutionary relation of both the pheromone and the wound response systems. In addition, the possible relation of this algal system and the wound healing reactions in higher plants deserves further investigation.