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A senescence-associated S-like RNase in the multicellular green alga Volvox carteri
Gene 274 (2001) 227–235 www.elsevier.com/locate/gene
A senescence-associated S-like RNase in the multicellular green alga Volvox carteri q Toshinobu Shimizu a, Tan Inoue a,b, Hideaki Shiraishi a,b,* a
b
Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
Received 9 April 2001; received in revised form 18 June 2001; accepted 6 July 2001 Received by D. Baulcombe
Abstract Asexual individuals of the green alga Volvox carteri consist of only two cell types: somatic and reproductive cells. The somatic cells are terminally differentiated, post-mitotic cells which undergo gradual senescence leading to cell death in every generation. To elucidate the selfdegrading process of macromolecules associated with senescence, we attempted to clone an RNase whose mRNA accumulation is increased during senescence. The corresponding cDNA clone VRN1, encoding an S-like RNase of V. carteri, is the first T2/S-like RNase to be cloned from green algae. Semi-quantitative RT-PCR analysis revealed that a relative amount of VRN1 mRNA is more than three-fold higher in the senescent somatic cells than in young somatic cells when the mRNA of ribosomal protein S18 is used as an internal standard. VRN1 mRNA is not induced by phosphate starvation, indicating that its accumulation during senescence is not due to a self-induced defect in utilizing phosphates. Similar regulation has been reported for RNS3, which encodes the S-like RNase that is induced in senescent leaves of Arabidopsis thaliana. These observations imply that VRN1 may promote RNA degradation during senescence of somatic cells in V. carteri, and that its regulation has similarity with that of certain senescence-associated RNases in higher plants. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Gene expression; Green algae; Phosphate starvation; RNA degradation
1. Introduction In the course of senescence, morphological and biochemical changes take place within cells. During leaf senescence in higher plants, dissociation of cellular organelles such as chloroplasts and degradation of macromolecules such as proteins and nucleic acids are observed (Thomas and Stoddart, 1980). The degradation of macromolecules is thought to be a process for redistributing limited nutrients from senescent organs to other developing parts of the plant. This is a genetically programmed process which requires
Abbreviations: cDNA, complementary DNA; GAL1, galactokinase; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RNase, ribonuclease; RT, reverse transcription; SDS, sodium dodecyl sulfate q The nucleotide sequence data reported have been deposited in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the Accession number AB034248. * Corresponding author. Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. Tel.: 181-75-753-3998; fax: 181-75-7533996. E-mail address: [email protected] (H. Shiraishi).
the expression of senescence-associated genes (Nooden and Guiamet, 1996). Though the physiology of senescence has been comparatively well studied in higher plants, that of green algae has not. From an evolutionary viewpoint, it is of interest to examine whether senescence in green algae shares common mechanisms with senescence in higher plants. Among green algae, Volvox carteri is especially suitable for studying senescence for the following reasons (Hagen and Kochert, 1980; Kirk, 1998). (1) One asexual life cycle of V. carteri is completed in only 48 h. (2) Asexual individuals consist of only two cell types: about 2000 biflagellate somatic cells and 8–16 reproductive cells (gonidia). (3) Its somatic cells are terminally differentiated, post-mitotic cells that undergo gradual senescence and eventual cell death in every generation. In addition, it has also been shown that the addition of cycloheximide, a translation inhibitor, prolongs the onset of senescence and delays the decline in somatic cell viability (Pommerville and Kochert, 1982), indicating that senescence in V. carteri is an active process which is genetically programmed and requires de novo protein translation. Although morphological and physiological changes in the
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00601-1
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somatic cells have been studied during the senescence of V. carteri (Pommerville and Kochert, 1981, 1982), molecular biological studies have not been reported, albeit that they are necessary for elucidating the regulation of senescenceassociated genes. To initiate such a study, we attempted to identify and characterize genes whose mRNA accumulation is increased during senescence in V. carteri. In this paper, we report the cloning and analysis of an RNase-encoding cDNA from V. carteri. This RNase, VRN1, belongs to the S-like RNase family, a subfamily of RNase T2. A relative amount of VRN1 mRNA is more than threefold higher in the senescent somatic cells than in young somatic cells when the mRNA of ribosomal protein S18 is used as an internal standard. In higher plants, RNases belonging to this family have been identified as members of senescence-associated RNases. Among these, RNS1, RNS2 and RNS3 of Arabidopsis thaliana are induced in leaves during senescence (Taylor et al., 1993), although RNS1 and RNS3 are up-regulated to a lesser extent than RNS2 (Bariola et al., 1994). In tomato, RNase LE and LX are induced during leaf senescence (Lers et al., 1998). Our finding that VRN1 is associated with senescence indicates that the regulation of S-like RNases during senescence is conserved among green algae and higher plants. With the exception of RNS3, plant S-like RNases are also known to be induced by phosphate starvation. We found that VRN1 is not induced by phosphate starvation, indicating that senescence and phosphate starvation do not fully share a common induction pathway in V. carteri. 2. Materials and methods 2.1. Strain and culture conditions Volvox carteri forma nagariensis (female strain HK10) was obtained from the University of Texas Culture Collection of Algae. Synchronous cultures were maintained in Standard Volvox Medium (SVM) (Kirk and Kirk, 1983) at 308C under a 16 h dark/32 h light (15,000 lux) cycle. 2.2. Preparation of stage- and cell type-specific RNA Cell types were separated and total RNA samples were isolated by the methods of Tam and Kirk (1991). Gonidial RNA was prepared from gonidia isolated just before the beginning of cleavage. Sheets of somatic cells embedded in extracellular matrix (ECM) were isolated from these same spheroids (which were by then 48 h old), incubated for another 24 h in SVM and then extracted to prepare the 72 h somatic cell (senescent somatic cell) RNA. For preparation of 24 h somatic cell RNA, newly inverted juveniles were isolated at the end of embryogenesis, incubated for 24 h in SVM and then used as the source of somatic cells for RNA extraction. To investigate the effect of phosphate starvation, 24 h spheroids enclosed by 72 h somatic cells were collected, washed extensively with phosphate-free
SVM and incubated in it for 24 h according to Hallmann (1999). RNAs from phosphate-deprived spheroids were then isolated. 2.3. Determination of somatic cell viability and preparation of chlorophyll The number of viable cells in a population was determined by Trypan Blue exclusion according to Pommerville and Kochert (1981). Chlorophyll was extracted and measured as described by Harris (1989). Values are means for three independent experiments. 2.4. Preparation of cellular extracts and analysis of RNA degrading activities Spheroids were collected by centrifugation. Pellets were suspended in a buffer containing 0.8 M Tris–Cl (pH 8.3), 0.4 M sucrose and 1% (w/v) 2-mercaptoethanol. Samples were frozen in liquid nitrogen and thawed in an ice bath. The cells were disrupted ultrasonically and lysates were centrifuged at 14,000 £ g for 20 min. The supernatants were frozen in liquid nitrogen and stored at 2808C. Protein concentrations of the solutions were determined by Bio-Rad Protein Assay (Bio-Rad). RNase activities of the cellular extracts of 24 and 72 h somatic cells were analyzed. The extracts were incubated in 0.3 M Tris–Cl (pH 6.8) at 308C with [a- 32P]GTPlabeled RNA prepared by transcription in vitro with T7 RNA polymerase. The DNA template for transcription was prepared by PCR with the primers T1 and T2 described in Section 2.5. The extent of RNA degradation was analyzed by polyacrylamide gel electrophoresis. The intensities of the bands were quantitated using a Bio-Image Analyzer BAS2500 (Fuji Film). 2.5. Oligonucleotides All the oligonucleotides used in this study were ordered from International Reagents Corporation. Sequences of the oligonucleotides were as follows: R1, 5 0 -ATH CAY GGN YTN TGG CC-3 0 ; R2, 5 0 -CAY GAG TGG NNN AAG CAY GGN ACN TG-3 0 ; V1, 5 0 -GTC ATT CCG CCC TTC AAG AGC CGT T-3 0 ; V2, 5 0 -ATT ATA TAC GCC TTC ATA CCC ACA A-3 0 ; S1, 5 0 -ATG GGC TCT CTG GTC CAC GGC G-3 0 ; S2, 5 0 -CGG ATC TTC TTC AGG CGC TCC A3 0 ; P1, 5 0 -CTG CCG GCA GTA GCA GCG CCA GGA A3 0 ; P2, 5 0 -CTA ATC CCA GGA CGA TAC GCC TGC T-3 0 ; A1, 5 0 -CAC GGA ATT CAA AAG AAT GAG ATT TCC TTC AAT-3 0 ; A2, 5 0 -TGA CAA TGG TCT TGC AGC TTC AGC CTC TCT-3 0 ; A3, 5 0 -AGA GAG GCT GAA GCT GCA AGA CCA TTG TCA TCC AT-3 0 ; A4, 5 0 -CAC GGA ATT CTA TAC CAT GGT CTC GTG CTT GA-3 0 ; A5, 5 0 -TCA TCC GCT CGA GTT ACA ATT TCC AAT CTA CGT GT-3 0 ; T1, 5 0 -CTA ATA CGA CTC ACT ATA GGG AGC AGC TGA TCT CCG GCA AGG A-3 0 ; T2, 5 0 CGT AGA TGG CCT CGT TAT CGA GCA-3 0 . R1 and R2 are degenerate primers that correspond to the C2 and C3
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regions of the S-like RNase family, respectively. They contain degenerate nucleotides: H, A/C/T; Y, C/T; or N, A/C/G/T. V1/V2, S1/S2 and P1/P2 are sense and antisense primers for VRN1, ribosomal protein S18 (Kirk et al., 1999) and extracellular phosphatase of Volvox (Hallmann, 1999), respectively. A1–5 are primers for construction of recombinant VRN1 designed to be expressed in Saccharomyces cerevisiae. T1/T2 are sense and antisense primers for alpha-1 tubulin exon 3 of Volvox (Mages et al., 1988). T1 contains the promoter sequence for T7 RNA polymerase. 2.6. cDNA cloning and sequence analysis Standard cloning techniques (Sambrook et al., 1989) were used throughout the study. Reverse transcription (RT) was performed using an RNA PCR Kit (AMV) Version 2.1 (Takara). Total RNA (200 ng) isolated from 72 h somatic cells was used as a template. First-strand cDNA was prepared using the oligo dT-adaptor primer from the kit. PCR amplification was carried out with Ex Taqe (Takara) according to the manufacturer’s instructions using 1% of the RT product as a template. Reactions consisted of 35 cycles of 948C for 30 s, 508C for 1 min and 728C for 2 min in a thermal cycler (Takara PCR thermal cycler MP). The first round of PCR was performed with R1 and oligo dT-adaptor primers. Second nested PCR was performed with R2 and oligo dT-adaptor primers. Reaction products were analyzed by agarose gel electrophoresis, and gel-purified DNA fragments were ligated into a vector, pBluescript II SK 1 (Stratagene). Recombinant plasmids were transformed into Escherichia coli strain JM109. Plasmids were analyzed by conventional techniques. Inserts were sequenced using a BcaBESTe Dideoxy Sequencing Kit (Takara) with an automated DNA sequencer (ALF express II, Amersham Pharmacia Biotech). To obtain full-length cDNA, the rapid amplification of cDNA ends (RACE) technique was performed using a 5 0 -Full RACE core set (Takara) according to the manufacturer’s instructions. To avoid errors in the nucleotide sequence that might be caused by the PCR reaction, DNA polymerase Ex Taqe (Takara), which possesses 3 0 to 5 0 exonuclease activity (proofreading activity), was used in all PCR reactions. In addition, clones from at least two independent reactions were examined to confirm that their sequences were identical. Sequence analysis was performed using the Gene Works computer program (Oxford Molecular Group Inc.). BLAST and FASTA were used for searching homologous sequences in the database (Altschul et al., 1990; Pearson and Lipman, 1988). The Nterminal signal sequence was predicted according to von Heijne (1986). 2.7. Southern blot hybridization DNA samples were separated on 1% (w/v) agarose gels and transferred to nylon membranes (Hybond-N, Amersham). The DNA fragment amplified by PCR with primers V1 and V2 was used as the probe for VRN1. DNA probes
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were labeled with [a- 32P]dCTP by using a BcaBESTe Labeling Kit (Takara). Prehybridizations and hybridizations were performed at 658C in 5 £ SSC, 0.1% (w/v) N-lauroylsarcosine-NaCl, 0.02% (w/v) SDS and 1% (w/v) blocking reagent (Boehringer Mannheim). The final washing condition for the blots was 1 £ SSC, 0.1% SDS at 658C. Blots were exposed to X-ray film at 2808C. 2.8. Semi-quantitative RT-PCR RT was performed by using an RNA PCR Kit (AMV) Version 2.1 (Takara). Total RNA (200 ng) isolated from gonidia, 24, 48 and 72 h somatic cells were used as templates. PCR was carried out as described in Section 2.6. The number of cycles was selected in each case such that the amount of the PCR products would be a linear reflection of the amount of template. Amplification of the VRN1 fragment was performed with primers V1 and V2. The reaction consisted of 27 cycles of 948C for 30 s, 588C for 30 s and 728C for 1 min. Amplification of the S18 fragment was performed with primers S1 and S2. The reaction consisted of 25 cycles of 948C for 30 s, 588C for 30 s and 728C for 1 min. Each reaction was performed seven times independently. To examine the effect of phosphate starvation, total RNA (200 ng) isolated from spheroids incubated with or without phosphate were used as templates. The fragment of extracellular phosphatase of Volvox was amplified with primers P1 and P2. The reaction consisted of 27 cycles of 948C for 30 s, 588C for 30 s and 728C for 1 min. Reaction products were analyzed by agarose gel electrophoresis, gels were stained with SYBR Green I Nucleic Acid Gel Stain (FMC), and the intensity of the bands was quantitated using a Fluor-S Multi Imager (Bio-Rad). 2.9. Western blotting The antiserum raised against the synthetic peptide was purchased from Takara. The sequence of the peptide, DLKAFDCDTSQEGNA, is identical to the deduced amino acid sequence of the VRN1 segment (amino acid positions 212–226). Cellular extracts were run on SDSpolyacrylamide gels and transferred to PVDF membranes (Hybond-P, Amersham). VRN1 was detected using an ECL Pluse Western blotting detection system (Amersham) according to the manufacturer’s instructions. 2.10. Expression of VRN1 in S. cerevisiae VRN1 cDNA was amplified by RT-PCR using RNA derived from 72 h somatic cells as a template, using primers A4 and A5. To obtain a modified VRN1 construct with a signal peptide of alpha factor from S. cerevisiae instead of the native VRN1 signal peptide, three PCRs were performed. First, a VRN1 cDNA without its signal sequence was amplified with primers A3 and A5. Then, the signal peptide of alpha factor was amplified from S. cerevisiae genomic DNA with primers A1 and A2. Since primers A2
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and A3 have complementary sequences, permitting the ends of these two DNA fragments to be annealed, the third PCR was performed to connect them using primers A1 and A5. The two cDNAs were inserted into the yeast expression vector pYES2 (Invitrogen) individually and the plasmids were introduced into yeast strain INVSc1 (Invitrogen) according to the instructions of the manufacturer. Transformants were selected on synthetic complete (SC) plates lacking uracil (SC 2 U plates). Since the cDNA inserts were under the control of the GAL1 promoter, recombinant VRN1 protein was induced by incubating the transformants in SC-U medium containing 2% galactose. RNase activities of yeast cell lysates were then assayed on RNase activity gels by the method of Yen and Green (1991).
3. Results 3.1. Senescence of somatic cells from V. carteri forma nagariensis
after cessation of cell division in forma weismannia (Pommerville and Kochert, 1982). We next examined the deterioration of the cellular function of somatic cells using chlorophyll content which is the most popular marker for plant cell senescence (Nooden and Guiamet, 1996). As shown in Fig. 1, the chlorophyll content of the somatic cells begins to decline prior to death. Together with the disorganization of chloroplast structure in senescent somatic cells (Pommerville and Kochert, 1981), it correlates with the deterioration of photosynthetic activities during senescence. We also found that the RNA content of somatic cells decreases during senescence (Fig. 1). Based on these observations, we decided to employ 72 h somatic cells as senescent somatic cells in forma nagariensis. At this time point, although somatic cells are fully viable, the cellular chlorophyll content and the amount of total RNAs have already begun to decline. 3.2. Cloning and sequence analysis of VRN1
In this study, we employed V. carteri forma nagariensis, a standard strain of V. carteri (Kirk, 1998), although previous studies of senescence have commonly used forma weismannia (Hagen and Kochert, 1980; Pommerville and Kochert, 1981, 1982). To employ forma nagariensis, we first determined the survival curve of its somatic cells by the dye exclusion method (Pommerville and Kochert, 1981, 1982). Somatic cells of V. carteri stop dividing before inversion and do not divide again (Kirk, 1998) so that their viability profiles were investigated from that time on. Survivorship curves for the somatic cells from forma nagariensis revealed that cell death begins at 96 h and is complete by 144 h (Fig. 1), in contrast to starting at 144 h and being completed by 192 h
As shown in Fig. 1, the RNA content of somatic cells from V. carteri declines before death. To examine whether RNase(s) are activated during senescence, the RNase activities of the cellular extracts from young somatic cells and those from senescent somatic cells were compared. The higher activities in the extracts from senescent somatic cells (Fig. 2) suggest the existence of senescence-associated RNase(s). Because S-like RNases that are widely conserved in eukaryotes are known to be associated with senescence in higher plants (Bariola and Green, 1997; Green, 1994), we attempted to clone the corresponding RNase from V. carteri. The cDNAs were prepared by RT using total RNA from 72 h senescent somatic cells as a template. We designed degenerate primers corresponding to the highly conserved
Fig. 1. Survival curves and changes in chlorophyll and RNA content of the somatic cells from V. carteri forma nagariensis. Viability was determined by selective incorporation of Trypan Blue into dead cells. The percentage of vital cells at each time point is shown. Total RNA and chlorophyll were extracted from the same quantities of somatic cells at each time point. Values are shown as a percentage of the maximum. Times indicated are after cessation of cell division.
Fig. 2. RNase activities in cellular extracts of V. carteri. Cellular extracts were prepared from young and senescent somatic cells. Extracts containing 1.5 mg of protein were incubated in 0.3 M Tris–Cl (pH 6.8) at 308C with [a- 32P]GTP-labeled RNA. The extent of RNA degradation was analyzed by polyacrylamide gel electrophoresis. The intensities of the bands were quantitated and relative intensities were plotted. Values are means ^ SE for three reactions.
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C2 and C3 regions of the S-like RNase family (Green, 1994). The fragment between C2 and the poly(A) tail was amplified first and then reamplified by nested PCR to obtain the fragment between C3 and the poly(A) tail. The resulting reaction products were cloned and their nucleotide sequences were determined. One of the most abundant fragments had features typical of S-like RNases in its nucleotide sequence, suggesting that it corresponded to a portion of the RNase cDNA. The rest of the cDNA was cloned by 5 0 RACE and the nucleotide sequence of the full-length cDNA was determined (Fig. 3). The cDNA termed as VRN1 has a putative polyadenylation signal characteristic of V. carteri, UGUAA (Kirk, 1998; Schmitt et al., 1992), 13 bp upstream from the poly(A) tail. The nucleotide sequence around the putative translation initiation codon ATG is ACCATGG, which conforms to the Kozak translation initiation consensus of eukaryotes (Kozak, 1986), and also with the consensus sequence
Fig. 4. Southern blot hybridization of V. carteri genomic DNA. The DNA (2 mg) was digested with PvuII (P), BglI (B) and EcoRV (E), and hybridized with the VRN1 probe.
found in Chlamydomonas (Ikeda and Miyasaka, 1998). An in-frame stop codon is present at nucleotide position 841 of the cDNA. The deduced protein consists of 256 amino acids with an estimated molecular weight of 29 kDa. The hydropathy plot of the protein reveals a highly hydrophobic Nterminus that is likely to be a secretory signal sequence. According to Southern blot analysis of genomic DNA from V. carteri probed with VRN1 cDNA, only one or two bands were detected with each restriction enzyme (Fig. 4), suggesting that close homologues of VRN1 are unlikely to exist in the genome of V. carteri. 3.3. Comparison of the sequence of VRN1 with other S-like RNases
Fig. 3. Sequence of VRN1 cDNA. The nucleotide and deduced amino acid sequences are shown. A putative polyadenylation signal UGUAA is underlined. Arrowheads indicate locations at which the putative N-terminal signal peptide is predicted to be cleaved.
The predicted amino acid sequence of VRN1 was compared with those of RNases belonging to the T2/S-like RNase family (Fig. 5). VRN1 is homologous with plant Slike RNases, especially with RNS3 from A. thaliana (38% amino acid identity). All five of the conserved regions (C1– C5) in the S-like RNase family (Green, 1994) are present in VRN1. Histidines at positions 76, 126 and 131, a glutamic acid at position 127 and a lysine at position 130 that are important for catalytic activity in RNase Rh from Rhizopus niveus (Irie, 1997) are conserved. Furthermore, the mature VRN1 without its putative N-terminal signal peptide contains eight conserved cysteines which may form disulfide bonds (Kurihara et al., 1992). VRN1 also possesses a Cterminal extension which does not generally exist in S-like RNases but is found in RNS2 from A. thaliana that is thought to be a vacuolar-targeting signal (Bariola et al., 1999; Taylor et al., 1993). Although the localization of VRN1 has not been determined, it is possible that the C-
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Fig. 5. Comparison of the deduced amino acid sequence of VRN1 with the following S-like RNase sequences: RNS1, 2 and 3 from A. thaliana (Bariola et al., 1994; Taylor et al., 1993), RNase LE and LX from tomato (Lycopersicon esculentum) (Jost et al., 1991; Loffler et al., 1993) and RNase PHYB from Physarum polycephalum (Inokuchi et al., 1993). Shaded boxes indicate conserved regions in the S-like RNase family (C1–C5) (Green, 1994). Asterisks indicate conserved cysteine residues.
terminal extension of VRN1 may also function as a vacuolar-targeting signal. 3.4. The expression of VRN1 To investigate the relationship between VRN1 and somatic cell senescence, its accumulation in young or senescent somatic cells was compared by semi-quantitative
RT-PCR analysis. The mRNA of ribosomal protein S18 was used as an internal standard, because it is highly expressed throughout the life cycle of V. carteri irrespective of the cell type and has been used as an internal standard in previous studies (Kirk et al., 1999). As shown in Fig. 6A, the relative abundance of VRN1 transcripts was more than three-fold higher in senescent somatic cells compared with young somatic cells. The rela-
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Fig. 6. Expression of VRN1 mRNA. (A) Semi-quantitative RT-PCR analysis of VRN1. Total RNA (200 ng) isolated from gonidia, 24, 48 and 72 h somatic cells were used as templates. Reaction products were analyzed by agarose gel electrophoresis (top). The intensities of the bands were quantitated and relative intensities were plotted (bottom). Values are means ^ SE for seven independent reactions normalized by the average value of S18 reaction products. (B) Semiquantitative RT-PCR analysis of VRN1 during phosphate starvation. Total RNA (200 ng) isolated from whole spheroids in the presence (lane 1) or absence (lane 2) of phosphates in the medium were used as templates. The fragments of VRN1, the extracellular phosphatase of Volvox and the ribosomal protein S18 were amplified. Reaction products were analyzed by agarose gel electrophoresis. The difference of the relative band intensities of VRN1 was less than 15% in three independent reactions. Extracellular phosphatase of Volvox is known to be induced during phosphate starvation (Hallmann, 1999).
tive amount is highest in senescent somatic cells although it is also expressed in gonidia. In higher plants, senescence-associated RNases are thought to degrade RNAs to remobilize phosphate. Most such RNases can be induced by phosphate starvation (Bariola and Green, 1997; Green, 1994). To examine
whether the expression of VRN1 is regulated by the external phosphate level, the amount of VRN1 mRNA was determined for spheroids that were suspended for 24 h either in normal phosphate-containing medium or in phosphatedeprived medium. As shown in Fig. 6B, expression levels were not affected by the depletion of phosphate, in contrast to that of the extracellular phosphatase of V. carteri which is induced by phosphate starvation (Hallmann, 1999). We also examined the expression of VRN1 protein by Western blotting analysis using antiserum raised against the VRN1 synthetic peptide. As shown in Fig. 7, the relative amount of VRN1 protein is increased in senescent somatic cells. The molecular mass of VRN1 protein was estimated as 26 kDa, which correlates well with the predicted molecular mass of 27 kDa without signal peptide. 3.5. Expression of recombinant VRN1 in S. cerevisiae
Fig. 7. Western blotting analysis of VRN1. Cellular extracts were prepared from young somatic cells (lane 1) and senescent somatic cells (lane 2). The extracts containing 15 mg of protein were electrophoresed in an SDS-polyacrylamide gel and probed with the VRN1 antiserum (left). Coomassiestained gel is shown for loading control (right).
To confirm the RNase activity of VRN1, recombinant proteins were expressed in S. cerevisiae. Two constructs for VRN1 recombinant proteins were employed, one consisting of the original VRN1 and one encoding a signal peptide of alpha factor from S. cerevisiae in place of the putative signal sequence (Brake et al., 1984). Both constructs were fused downstream of the GAL1 promoter. These plasmids and the vector pYES2 alone were separately transformed in yeast. The recombinant proteins were induced by incubating the transformants in galactosecontaining medium and the RNase activities were detected
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Fig. 8. Expression of recombinant VRN1 in S. cerevisiae. Recombinant VRN1 with the original signal peptide and another VRN1 construct with a signal peptide of alpha factor from S. cerevisiae were expressed under the control of GAL1 promoter in S. cerevisiae. Cell lysates were analyzed by RNase activity gels. Lane 1, vector control; lane 2, VRN1 with original signal peptide; lane 3, VRN1 with signal peptide of alpha factor.
as enzymatically active bands on RNase activity gels (Yen and Green, 1991). A band was observed with the recombinant VRN1 possessing the signal peptide of alpha factor when cellular extracts were examined (Fig. 8). For the original VRN1, a faint signal was also detected in the cellular extracts. These bands were not due to intrinsic RNases from yeast as judged by the control lane, indicating that VRN1 is indeed an active RNase.
4. Discussion In this study we cloned an RNase, VRN1, from the green alga V. carteri that is the first cDNA encoding a T2/S-like RNase to be identified in green algae. Our study shows that a relative amount of VRN1 mRNA is highest in senescent somatic cells and indicates that the regulation of VRN1 has similarity with certain senescence-associated RNases in higher plants. The self-degradation of macromolecules is frequently observed during senescence. We found that the RNA content of somatic cells from V. carteri declines prior to their death. The increase of the relative amount of VRN1 during senescence suggests that it may promote RNA degradation during senescence of somatic cells in V. carteri. However, moderate levels of expression of VRN1 are observed in young somatic cells and gonidia, suggesting that it also has constitutive roles in RNA metabolism. Because the RNA content of somatic cells sharply declines along with the progression of senescence, it is possible that the increase of the relative amount of VRN1 is not due to the up-regulation of its transcription but to the slower rate of its degradation compared with other transcripts. Although the
mechanism of the accumulation of VRN1 remains to be elucidated, our data indicate that the role of VRN1 in the RNA degradation programs might be more important in senescent somatic cells than in young cells. The majority of S-like RNases in higher plants are induced during both senescence and phosphate starvation. They are believed to be components of a phosphate remobilization system that recycles phosphates during senescence or phosphate starvation to supplement the limited supply of phosphate available from soil. In somatic cells of Volvox, cytoplasmic lipid bodies accumulate in the senescent cells (Pommerville and Kochert, 1981). Since the accumulation of lipid in algal cells can be a response to nutrient starvation (Healey, 1973), it was a possibility that phosphate starvation occurs during senescence, causing accumulation of VRN1. However, we confirmed that the expression of VRN1 is unaffected by phosphate depletions. S-like RNases that can be induced by either of the two stimuli have been found in higher plants. The expression of RNase NE of Nicotiana alata is not detected during leaf senescence and its response to phosphate limitation varies depending on tissue type (Dodds et al., 1996). RNS3 of A. thaliana is induced during senescence but not during phosphate starvation (Bariola et al., 1994). The regulation of such RNases indicates that senescence and phosphate starvation do not necessarily share the same signaling pathway. That the relative amount of VRN1 mRNA is increased during senescence while it is unaffected by depleting phosphates indicates that the increase during senescence is not related to the signaling pathway for phosphate starvation. Thus, regulation of VRN1 has some similarity with that of certain senescence-associated RNase(s) in higher plants such as RNS3. (Note. As an additional example for a different response to senescence and phosphate starvation in V. carteri, we found that the extracellular phosphatase of V. carteri is not induced during senescence (data not shown) although it is known to be induced during phosphate starvation (Hallmann, 1999).) Volvox is one of the genera belonging to the Volvocaceae. It is generally believed that multicellular species of volvocaceans have evolved from a Chlamydomonas-like unicellular ancestor along with multicellularity and cellular differentiation (Kirk, 1998; Schmitt et al., 1992). Because mortal somatic cells which undergo senescence also originated during this evolutional process, we think that Volvox provides an interesting system for studying the origin of senescence as well as the mechanism of senescence. The investigation of the genes from other volvocacean algae homologous with senescence-associated genes in Volvox would help in elucidating how their induction during senescence evolved.
Acknowledgements We thank Dr D.L. Kirk for kindly providing S18 cDNA
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and Dr K. Yasuda for his help in compiling data for the amino acid sequences of RNases. We also thank Dr R.T. Yu for critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas and a Grant-in-Aid for Exploratory Research from the Ministry of Education, Science, Sports and Culture of Japan.
References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Bariola, P.A., Green, P.J., 1997. Plant ribonucleases. In: D’Alessio, G., Riordan, J.F. (Eds.), Ribonucleases: Structures and Functions. Academic Press, New York, pp. 163–190. Bariola, P.A., Howard, C.J., Taylor, C.B., Verburg, M.T., Jaglan, V.D., Green, P.J., 1994. The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation. Plant J. 6, 673–685. Bariola, P.A., MacIntosh, G.C., Green, P.J., 1999. Regulation of S-like ribonuclease levels in Arabidopsis. Antisense inhibition of RNS1 or RNS2 elevates anthocyanin accumulation. Plant Physiol. 119, 331–342. Brake, A.J., Merryweather, J.P., Coit, D.G., Heberlein, U.A., Masiarz, F.R., Mullenbach, G.T., Urdea, M.S., Valenzuela, P., Barr, P.J., 1984. Alphafactor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 81, 4642–4646. Dodds, P.N., Clarke, A.E., Newbigin, E., 1996. Molecular characterisation of an S-like RNase of Nicotiana alata that is induced by phosphate starvation. Plant Mol. Biol. 31, 227–238. Green, P.J., 1994. The ribonucleases of higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 421–445. Hagen, G., Kochert, G., 1980. Protein synthesis in a new system for the study of senescence. Exp. Cell Res. 127, 451–457. Hallmann, A., 1999. Enzymes in the extracellular matrix of Volvox: an inducible, calcium-dependent phosphatase with a modular composition. J. Biol. Chem. 274, 1691–1697. Harris, E.H., 1989. The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego, CA. Healey, F.P., 1973. Inorganic nutrient uptake and deficiency in algae. CRC Crit. Rev. Microbiol. 3, 69–113. Ikeda, K., Miyasaka, H., 1998. Compilation of mRNA sequences surrounding the AUG translation initiation codon in the green alga Chlamydomonas reinhardtii. Biosci. Biotechnol. Biochem. 62, 2457–2459. Inokuchi, N., Koyama, T., Sawada, F., Irie, M., 1993. Purification, some properties, and primary structure of base non-specific ribonucleases from Physarum polycephalum. J. Biochem. (Tokyo) 113, 425–432. Irie, M., 1997. RNase T1/RNase T2 family RNases. In: D’Alessio, G., Riordan, J.F. (Eds.), Ribonucleases: Structures and Functions. Academic Press, New York, pp. 101–130. Jost, W., Bak, H., Glund, K., Terpstra, P., Beintema, J.J., 1991. Amino acid sequence of an extracellular, phosphate-starvation-induced ribonu-
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clease from cultured tomato (Lycopersicon esculentum) cells. Eur. J. Biochem. 198, 1–6. Kirk, D.L., 1998. Volvox: Molecular-Genetic Origins of Multicellularity and Cellular Differentiation, Cambridge University Press, Cambridge. Kirk, D.L., Kirk, M.M., 1983. Protein synthetic patterns during the asexual life cycle of Volvox carteri. Dev. Biol. 96, 493–506. Kirk, M.M., Stark, K., Miller, S.M., Muller, W., Taillon, B.E., Gruber, H., Schmitt, R., Kirk, D.L., 1999. regA, a Volvox gene that plays a central role in germ-soma differentiation, encodes a novel regulatory protein. Development 126, 639–647. Kozak, M., 1986. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283–292. Kurihara, H., Mitsui, Y., Ohgi, K., Irie, M., Mizuno, H., Nakamura, K.T., 1992. Crystal and molecular structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus. FEBS Lett. 306, 189– 192. Lers, A., Khalchitski, A., Lomaniec, E., Burd, S., Green, P.J., 1998. Senescence-induced RNases in tomato. Plant Mol. Biol. 36, 439–449. Loffler, A., Glund, K., Irie, M., 1993. Amino acid sequence of an intracellular, phosphate-starvation-induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells. Eur. J. Biochem. 214, 627–633. Mages, W., Salbaum, J.M., Harper, J.F., Schmitt, R., 1988. Organization and structure of Volvox a-tubulin genes. Mol. Gen. Genet. 213, 449– 458. Nooden, L.D., Guiamet, J.J., 1996. Genetic control of senescence and aging in plants. In: Schneider, E.L., Rowe, J.W. (Eds.), Handbook of the Biology of Aging, 4th Edition. Academic Press, New York, pp. 94–118. Pearson, W.R., Lipman, D.J., 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444–2448. Pommerville, J.C., Kochert, G.D., 1981. Changes in somatic cell structure during senescence of Volvox carteri. Eur. J. Cell Biol. 24, 236–243. Pommerville, J., Kochert, G., 1982. Effects of senescence on somatic cell physiology in the green alga Volvox carteri. Exp. Cell Res. 140, 39–45. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schmitt, R., Fabry, S., Kirk, D.L., 1992. In search of molecular origins of cellular differentiation in Volvox and its relatives. Int. Rev. Cytol. 139, 189–265. Tam, L.W., Kirk, D.L., 1991. Identification of cell-type-specific genes of Volvox carteri and characterization of their expression during the asexual life cycle. Dev. Biol. 145, 51–66. Taylor, C.B., Bariola, P.A., delCardayre, S.B., Raines, R.T., Green, P.J., 1993. RNS2: a senescence-associated RNase of Arabidopsis that diverged from the S-RNases before speciation. Proc. Natl. Acad. Sci. USA 90, 5118–5122. Thomas, H., Stoddart, J.L., 1980. Leaf senescence. Annu. Rev. Plant Physiol. 31, 83–111. von Heijne, G., 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683–4690. Yen, Y., Green, P.J., 1991. Identification and properties of the major ribonucleases of Arabidopsis thaliana. Plant Physiol. 97, 1487–1493.
A senescence-associated S-like RNase in the multicellular green alga Volvox carteri q Toshinobu Shimizu a, Tan Inoue a,b, Hideaki Shiraishi a,b,* a
b
Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
Received 9 April 2001; received in revised form 18 June 2001; accepted 6 July 2001 Received by D. Baulcombe
Abstract Asexual individuals of the green alga Volvox carteri consist of only two cell types: somatic and reproductive cells. The somatic cells are terminally differentiated, post-mitotic cells which undergo gradual senescence leading to cell death in every generation. To elucidate the selfdegrading process of macromolecules associated with senescence, we attempted to clone an RNase whose mRNA accumulation is increased during senescence. The corresponding cDNA clone VRN1, encoding an S-like RNase of V. carteri, is the first T2/S-like RNase to be cloned from green algae. Semi-quantitative RT-PCR analysis revealed that a relative amount of VRN1 mRNA is more than three-fold higher in the senescent somatic cells than in young somatic cells when the mRNA of ribosomal protein S18 is used as an internal standard. VRN1 mRNA is not induced by phosphate starvation, indicating that its accumulation during senescence is not due to a self-induced defect in utilizing phosphates. Similar regulation has been reported for RNS3, which encodes the S-like RNase that is induced in senescent leaves of Arabidopsis thaliana. These observations imply that VRN1 may promote RNA degradation during senescence of somatic cells in V. carteri, and that its regulation has similarity with that of certain senescence-associated RNases in higher plants. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Gene expression; Green algae; Phosphate starvation; RNA degradation
1. Introduction In the course of senescence, morphological and biochemical changes take place within cells. During leaf senescence in higher plants, dissociation of cellular organelles such as chloroplasts and degradation of macromolecules such as proteins and nucleic acids are observed (Thomas and Stoddart, 1980). The degradation of macromolecules is thought to be a process for redistributing limited nutrients from senescent organs to other developing parts of the plant. This is a genetically programmed process which requires
Abbreviations: cDNA, complementary DNA; GAL1, galactokinase; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RNase, ribonuclease; RT, reverse transcription; SDS, sodium dodecyl sulfate q The nucleotide sequence data reported have been deposited in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the Accession number AB034248. * Corresponding author. Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. Tel.: 181-75-753-3998; fax: 181-75-7533996. E-mail address: [email protected] (H. Shiraishi).
the expression of senescence-associated genes (Nooden and Guiamet, 1996). Though the physiology of senescence has been comparatively well studied in higher plants, that of green algae has not. From an evolutionary viewpoint, it is of interest to examine whether senescence in green algae shares common mechanisms with senescence in higher plants. Among green algae, Volvox carteri is especially suitable for studying senescence for the following reasons (Hagen and Kochert, 1980; Kirk, 1998). (1) One asexual life cycle of V. carteri is completed in only 48 h. (2) Asexual individuals consist of only two cell types: about 2000 biflagellate somatic cells and 8–16 reproductive cells (gonidia). (3) Its somatic cells are terminally differentiated, post-mitotic cells that undergo gradual senescence and eventual cell death in every generation. In addition, it has also been shown that the addition of cycloheximide, a translation inhibitor, prolongs the onset of senescence and delays the decline in somatic cell viability (Pommerville and Kochert, 1982), indicating that senescence in V. carteri is an active process which is genetically programmed and requires de novo protein translation. Although morphological and physiological changes in the
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00601-1
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somatic cells have been studied during the senescence of V. carteri (Pommerville and Kochert, 1981, 1982), molecular biological studies have not been reported, albeit that they are necessary for elucidating the regulation of senescenceassociated genes. To initiate such a study, we attempted to identify and characterize genes whose mRNA accumulation is increased during senescence in V. carteri. In this paper, we report the cloning and analysis of an RNase-encoding cDNA from V. carteri. This RNase, VRN1, belongs to the S-like RNase family, a subfamily of RNase T2. A relative amount of VRN1 mRNA is more than threefold higher in the senescent somatic cells than in young somatic cells when the mRNA of ribosomal protein S18 is used as an internal standard. In higher plants, RNases belonging to this family have been identified as members of senescence-associated RNases. Among these, RNS1, RNS2 and RNS3 of Arabidopsis thaliana are induced in leaves during senescence (Taylor et al., 1993), although RNS1 and RNS3 are up-regulated to a lesser extent than RNS2 (Bariola et al., 1994). In tomato, RNase LE and LX are induced during leaf senescence (Lers et al., 1998). Our finding that VRN1 is associated with senescence indicates that the regulation of S-like RNases during senescence is conserved among green algae and higher plants. With the exception of RNS3, plant S-like RNases are also known to be induced by phosphate starvation. We found that VRN1 is not induced by phosphate starvation, indicating that senescence and phosphate starvation do not fully share a common induction pathway in V. carteri. 2. Materials and methods 2.1. Strain and culture conditions Volvox carteri forma nagariensis (female strain HK10) was obtained from the University of Texas Culture Collection of Algae. Synchronous cultures were maintained in Standard Volvox Medium (SVM) (Kirk and Kirk, 1983) at 308C under a 16 h dark/32 h light (15,000 lux) cycle. 2.2. Preparation of stage- and cell type-specific RNA Cell types were separated and total RNA samples were isolated by the methods of Tam and Kirk (1991). Gonidial RNA was prepared from gonidia isolated just before the beginning of cleavage. Sheets of somatic cells embedded in extracellular matrix (ECM) were isolated from these same spheroids (which were by then 48 h old), incubated for another 24 h in SVM and then extracted to prepare the 72 h somatic cell (senescent somatic cell) RNA. For preparation of 24 h somatic cell RNA, newly inverted juveniles were isolated at the end of embryogenesis, incubated for 24 h in SVM and then used as the source of somatic cells for RNA extraction. To investigate the effect of phosphate starvation, 24 h spheroids enclosed by 72 h somatic cells were collected, washed extensively with phosphate-free
SVM and incubated in it for 24 h according to Hallmann (1999). RNAs from phosphate-deprived spheroids were then isolated. 2.3. Determination of somatic cell viability and preparation of chlorophyll The number of viable cells in a population was determined by Trypan Blue exclusion according to Pommerville and Kochert (1981). Chlorophyll was extracted and measured as described by Harris (1989). Values are means for three independent experiments. 2.4. Preparation of cellular extracts and analysis of RNA degrading activities Spheroids were collected by centrifugation. Pellets were suspended in a buffer containing 0.8 M Tris–Cl (pH 8.3), 0.4 M sucrose and 1% (w/v) 2-mercaptoethanol. Samples were frozen in liquid nitrogen and thawed in an ice bath. The cells were disrupted ultrasonically and lysates were centrifuged at 14,000 £ g for 20 min. The supernatants were frozen in liquid nitrogen and stored at 2808C. Protein concentrations of the solutions were determined by Bio-Rad Protein Assay (Bio-Rad). RNase activities of the cellular extracts of 24 and 72 h somatic cells were analyzed. The extracts were incubated in 0.3 M Tris–Cl (pH 6.8) at 308C with [a- 32P]GTPlabeled RNA prepared by transcription in vitro with T7 RNA polymerase. The DNA template for transcription was prepared by PCR with the primers T1 and T2 described in Section 2.5. The extent of RNA degradation was analyzed by polyacrylamide gel electrophoresis. The intensities of the bands were quantitated using a Bio-Image Analyzer BAS2500 (Fuji Film). 2.5. Oligonucleotides All the oligonucleotides used in this study were ordered from International Reagents Corporation. Sequences of the oligonucleotides were as follows: R1, 5 0 -ATH CAY GGN YTN TGG CC-3 0 ; R2, 5 0 -CAY GAG TGG NNN AAG CAY GGN ACN TG-3 0 ; V1, 5 0 -GTC ATT CCG CCC TTC AAG AGC CGT T-3 0 ; V2, 5 0 -ATT ATA TAC GCC TTC ATA CCC ACA A-3 0 ; S1, 5 0 -ATG GGC TCT CTG GTC CAC GGC G-3 0 ; S2, 5 0 -CGG ATC TTC TTC AGG CGC TCC A3 0 ; P1, 5 0 -CTG CCG GCA GTA GCA GCG CCA GGA A3 0 ; P2, 5 0 -CTA ATC CCA GGA CGA TAC GCC TGC T-3 0 ; A1, 5 0 -CAC GGA ATT CAA AAG AAT GAG ATT TCC TTC AAT-3 0 ; A2, 5 0 -TGA CAA TGG TCT TGC AGC TTC AGC CTC TCT-3 0 ; A3, 5 0 -AGA GAG GCT GAA GCT GCA AGA CCA TTG TCA TCC AT-3 0 ; A4, 5 0 -CAC GGA ATT CTA TAC CAT GGT CTC GTG CTT GA-3 0 ; A5, 5 0 -TCA TCC GCT CGA GTT ACA ATT TCC AAT CTA CGT GT-3 0 ; T1, 5 0 -CTA ATA CGA CTC ACT ATA GGG AGC AGC TGA TCT CCG GCA AGG A-3 0 ; T2, 5 0 CGT AGA TGG CCT CGT TAT CGA GCA-3 0 . R1 and R2 are degenerate primers that correspond to the C2 and C3
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regions of the S-like RNase family, respectively. They contain degenerate nucleotides: H, A/C/T; Y, C/T; or N, A/C/G/T. V1/V2, S1/S2 and P1/P2 are sense and antisense primers for VRN1, ribosomal protein S18 (Kirk et al., 1999) and extracellular phosphatase of Volvox (Hallmann, 1999), respectively. A1–5 are primers for construction of recombinant VRN1 designed to be expressed in Saccharomyces cerevisiae. T1/T2 are sense and antisense primers for alpha-1 tubulin exon 3 of Volvox (Mages et al., 1988). T1 contains the promoter sequence for T7 RNA polymerase. 2.6. cDNA cloning and sequence analysis Standard cloning techniques (Sambrook et al., 1989) were used throughout the study. Reverse transcription (RT) was performed using an RNA PCR Kit (AMV) Version 2.1 (Takara). Total RNA (200 ng) isolated from 72 h somatic cells was used as a template. First-strand cDNA was prepared using the oligo dT-adaptor primer from the kit. PCR amplification was carried out with Ex Taqe (Takara) according to the manufacturer’s instructions using 1% of the RT product as a template. Reactions consisted of 35 cycles of 948C for 30 s, 508C for 1 min and 728C for 2 min in a thermal cycler (Takara PCR thermal cycler MP). The first round of PCR was performed with R1 and oligo dT-adaptor primers. Second nested PCR was performed with R2 and oligo dT-adaptor primers. Reaction products were analyzed by agarose gel electrophoresis, and gel-purified DNA fragments were ligated into a vector, pBluescript II SK 1 (Stratagene). Recombinant plasmids were transformed into Escherichia coli strain JM109. Plasmids were analyzed by conventional techniques. Inserts were sequenced using a BcaBESTe Dideoxy Sequencing Kit (Takara) with an automated DNA sequencer (ALF express II, Amersham Pharmacia Biotech). To obtain full-length cDNA, the rapid amplification of cDNA ends (RACE) technique was performed using a 5 0 -Full RACE core set (Takara) according to the manufacturer’s instructions. To avoid errors in the nucleotide sequence that might be caused by the PCR reaction, DNA polymerase Ex Taqe (Takara), which possesses 3 0 to 5 0 exonuclease activity (proofreading activity), was used in all PCR reactions. In addition, clones from at least two independent reactions were examined to confirm that their sequences were identical. Sequence analysis was performed using the Gene Works computer program (Oxford Molecular Group Inc.). BLAST and FASTA were used for searching homologous sequences in the database (Altschul et al., 1990; Pearson and Lipman, 1988). The Nterminal signal sequence was predicted according to von Heijne (1986). 2.7. Southern blot hybridization DNA samples were separated on 1% (w/v) agarose gels and transferred to nylon membranes (Hybond-N, Amersham). The DNA fragment amplified by PCR with primers V1 and V2 was used as the probe for VRN1. DNA probes
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were labeled with [a- 32P]dCTP by using a BcaBESTe Labeling Kit (Takara). Prehybridizations and hybridizations were performed at 658C in 5 £ SSC, 0.1% (w/v) N-lauroylsarcosine-NaCl, 0.02% (w/v) SDS and 1% (w/v) blocking reagent (Boehringer Mannheim). The final washing condition for the blots was 1 £ SSC, 0.1% SDS at 658C. Blots were exposed to X-ray film at 2808C. 2.8. Semi-quantitative RT-PCR RT was performed by using an RNA PCR Kit (AMV) Version 2.1 (Takara). Total RNA (200 ng) isolated from gonidia, 24, 48 and 72 h somatic cells were used as templates. PCR was carried out as described in Section 2.6. The number of cycles was selected in each case such that the amount of the PCR products would be a linear reflection of the amount of template. Amplification of the VRN1 fragment was performed with primers V1 and V2. The reaction consisted of 27 cycles of 948C for 30 s, 588C for 30 s and 728C for 1 min. Amplification of the S18 fragment was performed with primers S1 and S2. The reaction consisted of 25 cycles of 948C for 30 s, 588C for 30 s and 728C for 1 min. Each reaction was performed seven times independently. To examine the effect of phosphate starvation, total RNA (200 ng) isolated from spheroids incubated with or without phosphate were used as templates. The fragment of extracellular phosphatase of Volvox was amplified with primers P1 and P2. The reaction consisted of 27 cycles of 948C for 30 s, 588C for 30 s and 728C for 1 min. Reaction products were analyzed by agarose gel electrophoresis, gels were stained with SYBR Green I Nucleic Acid Gel Stain (FMC), and the intensity of the bands was quantitated using a Fluor-S Multi Imager (Bio-Rad). 2.9. Western blotting The antiserum raised against the synthetic peptide was purchased from Takara. The sequence of the peptide, DLKAFDCDTSQEGNA, is identical to the deduced amino acid sequence of the VRN1 segment (amino acid positions 212–226). Cellular extracts were run on SDSpolyacrylamide gels and transferred to PVDF membranes (Hybond-P, Amersham). VRN1 was detected using an ECL Pluse Western blotting detection system (Amersham) according to the manufacturer’s instructions. 2.10. Expression of VRN1 in S. cerevisiae VRN1 cDNA was amplified by RT-PCR using RNA derived from 72 h somatic cells as a template, using primers A4 and A5. To obtain a modified VRN1 construct with a signal peptide of alpha factor from S. cerevisiae instead of the native VRN1 signal peptide, three PCRs were performed. First, a VRN1 cDNA without its signal sequence was amplified with primers A3 and A5. Then, the signal peptide of alpha factor was amplified from S. cerevisiae genomic DNA with primers A1 and A2. Since primers A2
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and A3 have complementary sequences, permitting the ends of these two DNA fragments to be annealed, the third PCR was performed to connect them using primers A1 and A5. The two cDNAs were inserted into the yeast expression vector pYES2 (Invitrogen) individually and the plasmids were introduced into yeast strain INVSc1 (Invitrogen) according to the instructions of the manufacturer. Transformants were selected on synthetic complete (SC) plates lacking uracil (SC 2 U plates). Since the cDNA inserts were under the control of the GAL1 promoter, recombinant VRN1 protein was induced by incubating the transformants in SC-U medium containing 2% galactose. RNase activities of yeast cell lysates were then assayed on RNase activity gels by the method of Yen and Green (1991).
3. Results 3.1. Senescence of somatic cells from V. carteri forma nagariensis
after cessation of cell division in forma weismannia (Pommerville and Kochert, 1982). We next examined the deterioration of the cellular function of somatic cells using chlorophyll content which is the most popular marker for plant cell senescence (Nooden and Guiamet, 1996). As shown in Fig. 1, the chlorophyll content of the somatic cells begins to decline prior to death. Together with the disorganization of chloroplast structure in senescent somatic cells (Pommerville and Kochert, 1981), it correlates with the deterioration of photosynthetic activities during senescence. We also found that the RNA content of somatic cells decreases during senescence (Fig. 1). Based on these observations, we decided to employ 72 h somatic cells as senescent somatic cells in forma nagariensis. At this time point, although somatic cells are fully viable, the cellular chlorophyll content and the amount of total RNAs have already begun to decline. 3.2. Cloning and sequence analysis of VRN1
In this study, we employed V. carteri forma nagariensis, a standard strain of V. carteri (Kirk, 1998), although previous studies of senescence have commonly used forma weismannia (Hagen and Kochert, 1980; Pommerville and Kochert, 1981, 1982). To employ forma nagariensis, we first determined the survival curve of its somatic cells by the dye exclusion method (Pommerville and Kochert, 1981, 1982). Somatic cells of V. carteri stop dividing before inversion and do not divide again (Kirk, 1998) so that their viability profiles were investigated from that time on. Survivorship curves for the somatic cells from forma nagariensis revealed that cell death begins at 96 h and is complete by 144 h (Fig. 1), in contrast to starting at 144 h and being completed by 192 h
As shown in Fig. 1, the RNA content of somatic cells from V. carteri declines before death. To examine whether RNase(s) are activated during senescence, the RNase activities of the cellular extracts from young somatic cells and those from senescent somatic cells were compared. The higher activities in the extracts from senescent somatic cells (Fig. 2) suggest the existence of senescence-associated RNase(s). Because S-like RNases that are widely conserved in eukaryotes are known to be associated with senescence in higher plants (Bariola and Green, 1997; Green, 1994), we attempted to clone the corresponding RNase from V. carteri. The cDNAs were prepared by RT using total RNA from 72 h senescent somatic cells as a template. We designed degenerate primers corresponding to the highly conserved
Fig. 1. Survival curves and changes in chlorophyll and RNA content of the somatic cells from V. carteri forma nagariensis. Viability was determined by selective incorporation of Trypan Blue into dead cells. The percentage of vital cells at each time point is shown. Total RNA and chlorophyll were extracted from the same quantities of somatic cells at each time point. Values are shown as a percentage of the maximum. Times indicated are after cessation of cell division.
Fig. 2. RNase activities in cellular extracts of V. carteri. Cellular extracts were prepared from young and senescent somatic cells. Extracts containing 1.5 mg of protein were incubated in 0.3 M Tris–Cl (pH 6.8) at 308C with [a- 32P]GTP-labeled RNA. The extent of RNA degradation was analyzed by polyacrylamide gel electrophoresis. The intensities of the bands were quantitated and relative intensities were plotted. Values are means ^ SE for three reactions.
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C2 and C3 regions of the S-like RNase family (Green, 1994). The fragment between C2 and the poly(A) tail was amplified first and then reamplified by nested PCR to obtain the fragment between C3 and the poly(A) tail. The resulting reaction products were cloned and their nucleotide sequences were determined. One of the most abundant fragments had features typical of S-like RNases in its nucleotide sequence, suggesting that it corresponded to a portion of the RNase cDNA. The rest of the cDNA was cloned by 5 0 RACE and the nucleotide sequence of the full-length cDNA was determined (Fig. 3). The cDNA termed as VRN1 has a putative polyadenylation signal characteristic of V. carteri, UGUAA (Kirk, 1998; Schmitt et al., 1992), 13 bp upstream from the poly(A) tail. The nucleotide sequence around the putative translation initiation codon ATG is ACCATGG, which conforms to the Kozak translation initiation consensus of eukaryotes (Kozak, 1986), and also with the consensus sequence
Fig. 4. Southern blot hybridization of V. carteri genomic DNA. The DNA (2 mg) was digested with PvuII (P), BglI (B) and EcoRV (E), and hybridized with the VRN1 probe.
found in Chlamydomonas (Ikeda and Miyasaka, 1998). An in-frame stop codon is present at nucleotide position 841 of the cDNA. The deduced protein consists of 256 amino acids with an estimated molecular weight of 29 kDa. The hydropathy plot of the protein reveals a highly hydrophobic Nterminus that is likely to be a secretory signal sequence. According to Southern blot analysis of genomic DNA from V. carteri probed with VRN1 cDNA, only one or two bands were detected with each restriction enzyme (Fig. 4), suggesting that close homologues of VRN1 are unlikely to exist in the genome of V. carteri. 3.3. Comparison of the sequence of VRN1 with other S-like RNases
Fig. 3. Sequence of VRN1 cDNA. The nucleotide and deduced amino acid sequences are shown. A putative polyadenylation signal UGUAA is underlined. Arrowheads indicate locations at which the putative N-terminal signal peptide is predicted to be cleaved.
The predicted amino acid sequence of VRN1 was compared with those of RNases belonging to the T2/S-like RNase family (Fig. 5). VRN1 is homologous with plant Slike RNases, especially with RNS3 from A. thaliana (38% amino acid identity). All five of the conserved regions (C1– C5) in the S-like RNase family (Green, 1994) are present in VRN1. Histidines at positions 76, 126 and 131, a glutamic acid at position 127 and a lysine at position 130 that are important for catalytic activity in RNase Rh from Rhizopus niveus (Irie, 1997) are conserved. Furthermore, the mature VRN1 without its putative N-terminal signal peptide contains eight conserved cysteines which may form disulfide bonds (Kurihara et al., 1992). VRN1 also possesses a Cterminal extension which does not generally exist in S-like RNases but is found in RNS2 from A. thaliana that is thought to be a vacuolar-targeting signal (Bariola et al., 1999; Taylor et al., 1993). Although the localization of VRN1 has not been determined, it is possible that the C-
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Fig. 5. Comparison of the deduced amino acid sequence of VRN1 with the following S-like RNase sequences: RNS1, 2 and 3 from A. thaliana (Bariola et al., 1994; Taylor et al., 1993), RNase LE and LX from tomato (Lycopersicon esculentum) (Jost et al., 1991; Loffler et al., 1993) and RNase PHYB from Physarum polycephalum (Inokuchi et al., 1993). Shaded boxes indicate conserved regions in the S-like RNase family (C1–C5) (Green, 1994). Asterisks indicate conserved cysteine residues.
terminal extension of VRN1 may also function as a vacuolar-targeting signal. 3.4. The expression of VRN1 To investigate the relationship between VRN1 and somatic cell senescence, its accumulation in young or senescent somatic cells was compared by semi-quantitative
RT-PCR analysis. The mRNA of ribosomal protein S18 was used as an internal standard, because it is highly expressed throughout the life cycle of V. carteri irrespective of the cell type and has been used as an internal standard in previous studies (Kirk et al., 1999). As shown in Fig. 6A, the relative abundance of VRN1 transcripts was more than three-fold higher in senescent somatic cells compared with young somatic cells. The rela-
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Fig. 6. Expression of VRN1 mRNA. (A) Semi-quantitative RT-PCR analysis of VRN1. Total RNA (200 ng) isolated from gonidia, 24, 48 and 72 h somatic cells were used as templates. Reaction products were analyzed by agarose gel electrophoresis (top). The intensities of the bands were quantitated and relative intensities were plotted (bottom). Values are means ^ SE for seven independent reactions normalized by the average value of S18 reaction products. (B) Semiquantitative RT-PCR analysis of VRN1 during phosphate starvation. Total RNA (200 ng) isolated from whole spheroids in the presence (lane 1) or absence (lane 2) of phosphates in the medium were used as templates. The fragments of VRN1, the extracellular phosphatase of Volvox and the ribosomal protein S18 were amplified. Reaction products were analyzed by agarose gel electrophoresis. The difference of the relative band intensities of VRN1 was less than 15% in three independent reactions. Extracellular phosphatase of Volvox is known to be induced during phosphate starvation (Hallmann, 1999).
tive amount is highest in senescent somatic cells although it is also expressed in gonidia. In higher plants, senescence-associated RNases are thought to degrade RNAs to remobilize phosphate. Most such RNases can be induced by phosphate starvation (Bariola and Green, 1997; Green, 1994). To examine
whether the expression of VRN1 is regulated by the external phosphate level, the amount of VRN1 mRNA was determined for spheroids that were suspended for 24 h either in normal phosphate-containing medium or in phosphatedeprived medium. As shown in Fig. 6B, expression levels were not affected by the depletion of phosphate, in contrast to that of the extracellular phosphatase of V. carteri which is induced by phosphate starvation (Hallmann, 1999). We also examined the expression of VRN1 protein by Western blotting analysis using antiserum raised against the VRN1 synthetic peptide. As shown in Fig. 7, the relative amount of VRN1 protein is increased in senescent somatic cells. The molecular mass of VRN1 protein was estimated as 26 kDa, which correlates well with the predicted molecular mass of 27 kDa without signal peptide. 3.5. Expression of recombinant VRN1 in S. cerevisiae
Fig. 7. Western blotting analysis of VRN1. Cellular extracts were prepared from young somatic cells (lane 1) and senescent somatic cells (lane 2). The extracts containing 15 mg of protein were electrophoresed in an SDS-polyacrylamide gel and probed with the VRN1 antiserum (left). Coomassiestained gel is shown for loading control (right).
To confirm the RNase activity of VRN1, recombinant proteins were expressed in S. cerevisiae. Two constructs for VRN1 recombinant proteins were employed, one consisting of the original VRN1 and one encoding a signal peptide of alpha factor from S. cerevisiae in place of the putative signal sequence (Brake et al., 1984). Both constructs were fused downstream of the GAL1 promoter. These plasmids and the vector pYES2 alone were separately transformed in yeast. The recombinant proteins were induced by incubating the transformants in galactosecontaining medium and the RNase activities were detected
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Fig. 8. Expression of recombinant VRN1 in S. cerevisiae. Recombinant VRN1 with the original signal peptide and another VRN1 construct with a signal peptide of alpha factor from S. cerevisiae were expressed under the control of GAL1 promoter in S. cerevisiae. Cell lysates were analyzed by RNase activity gels. Lane 1, vector control; lane 2, VRN1 with original signal peptide; lane 3, VRN1 with signal peptide of alpha factor.
as enzymatically active bands on RNase activity gels (Yen and Green, 1991). A band was observed with the recombinant VRN1 possessing the signal peptide of alpha factor when cellular extracts were examined (Fig. 8). For the original VRN1, a faint signal was also detected in the cellular extracts. These bands were not due to intrinsic RNases from yeast as judged by the control lane, indicating that VRN1 is indeed an active RNase.
4. Discussion In this study we cloned an RNase, VRN1, from the green alga V. carteri that is the first cDNA encoding a T2/S-like RNase to be identified in green algae. Our study shows that a relative amount of VRN1 mRNA is highest in senescent somatic cells and indicates that the regulation of VRN1 has similarity with certain senescence-associated RNases in higher plants. The self-degradation of macromolecules is frequently observed during senescence. We found that the RNA content of somatic cells from V. carteri declines prior to their death. The increase of the relative amount of VRN1 during senescence suggests that it may promote RNA degradation during senescence of somatic cells in V. carteri. However, moderate levels of expression of VRN1 are observed in young somatic cells and gonidia, suggesting that it also has constitutive roles in RNA metabolism. Because the RNA content of somatic cells sharply declines along with the progression of senescence, it is possible that the increase of the relative amount of VRN1 is not due to the up-regulation of its transcription but to the slower rate of its degradation compared with other transcripts. Although the
mechanism of the accumulation of VRN1 remains to be elucidated, our data indicate that the role of VRN1 in the RNA degradation programs might be more important in senescent somatic cells than in young cells. The majority of S-like RNases in higher plants are induced during both senescence and phosphate starvation. They are believed to be components of a phosphate remobilization system that recycles phosphates during senescence or phosphate starvation to supplement the limited supply of phosphate available from soil. In somatic cells of Volvox, cytoplasmic lipid bodies accumulate in the senescent cells (Pommerville and Kochert, 1981). Since the accumulation of lipid in algal cells can be a response to nutrient starvation (Healey, 1973), it was a possibility that phosphate starvation occurs during senescence, causing accumulation of VRN1. However, we confirmed that the expression of VRN1 is unaffected by phosphate depletions. S-like RNases that can be induced by either of the two stimuli have been found in higher plants. The expression of RNase NE of Nicotiana alata is not detected during leaf senescence and its response to phosphate limitation varies depending on tissue type (Dodds et al., 1996). RNS3 of A. thaliana is induced during senescence but not during phosphate starvation (Bariola et al., 1994). The regulation of such RNases indicates that senescence and phosphate starvation do not necessarily share the same signaling pathway. That the relative amount of VRN1 mRNA is increased during senescence while it is unaffected by depleting phosphates indicates that the increase during senescence is not related to the signaling pathway for phosphate starvation. Thus, regulation of VRN1 has some similarity with that of certain senescence-associated RNase(s) in higher plants such as RNS3. (Note. As an additional example for a different response to senescence and phosphate starvation in V. carteri, we found that the extracellular phosphatase of V. carteri is not induced during senescence (data not shown) although it is known to be induced during phosphate starvation (Hallmann, 1999).) Volvox is one of the genera belonging to the Volvocaceae. It is generally believed that multicellular species of volvocaceans have evolved from a Chlamydomonas-like unicellular ancestor along with multicellularity and cellular differentiation (Kirk, 1998; Schmitt et al., 1992). Because mortal somatic cells which undergo senescence also originated during this evolutional process, we think that Volvox provides an interesting system for studying the origin of senescence as well as the mechanism of senescence. The investigation of the genes from other volvocacean algae homologous with senescence-associated genes in Volvox would help in elucidating how their induction during senescence evolved.
Acknowledgements We thank Dr D.L. Kirk for kindly providing S18 cDNA
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and Dr K. Yasuda for his help in compiling data for the amino acid sequences of RNases. We also thank Dr R.T. Yu for critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas and a Grant-in-Aid for Exploratory Research from the Ministry of Education, Science, Sports and Culture of Japan.
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