Proc. Nati. Acad. Sci. USA Vol. 87, pp. 195-199, January 1990 Botany

Defective chlorophyll a/b-binding protein genes in the genome of a homosporous fern E. PICHERSKY*t, D. SOLTIS$, AND P. SOLTISf *Department of Biology, University of Michigan, Ann Arbor, Ml 48109; and tDepartment of Botany, Washington State University, Pullman, WA 99164 Communicated by Warren H. Wagner, October 13, 1989

ABSTRACT The majority of homosporous ferns have a revealed that these six branches ofthe CAB gene family arose chromosome number that is severalfold greater than that of by gene duplication and divergence prior to the radiation of diploid seed plants. These fern species have therefore been the angiosperms. Within each branch of the CAB family, generally considered to be of polyploid origin. Enzyme elec- more recent duplications have also occurred, so that the same trophoretic investigations have demonstrated, however, that type ofCAB polypeptide is sometimes encoded by more than within fern genera, species having the lowest chromosome one gene. Unlike the situation found in many gene families in numbers (n = 27-52) have the number of isozymes typical of animals, the CAB gene family in all angiosperm genomes so diploid seed plants; there is no isozyme evidence for polyploidy far examined does not contain many defective genes (a of these plants. We have constructed a genomic DNA library defective gene is defined here as a gene containing a struc- from Polystichum munitum (n = 41), a homosporous fern, and tural defect that either prevents its transcription or results in have screened the library for sequences homologous to the no functional protein issuing from the mRNA). Indeed, to chlorophyll a/b-binding (CAB) protein genes of higher plants. date only a single CAB gene known to be defective has been The majority of the sequences isolated and characterized by found (9) out ofthe many scores ofCAB genes analyzed. This nucleotide sequence determination represent defective CAB is also the case in another gene family extensively analyzed genes. This result is in contrast to the situation in the genomes in higher plants, the RBCS gene family encoding the small of diploid angiosperms, where most, and sometimes all, copies subunits of ribulose-bisphosphate carboxylase; only a few of the CAB gene family represent functional members. Several defective RBCS genes have so far been found (10). Although hypotheses could explain the existence of multiple defective other gene families in higher plants have not been as exten- CAB genes in P. munitum. (i) The defective CAB genes are the sively analyzed as the CAB and RBCS genes families, result of "gene silencing" following polyploidy. (u) P. munitum multiple defective genes have not yet been encountered. has not gone through a polyploidization event, but several, and In this report we describe the characterization of several perhaps the majority, of its CAB genes have mutated to a genes encoding CAB proteins from the genome of the ho- nonfunctional state (a phenomenon not yet observed in any of mosporous fern Polystichum munitum (n = 41, ref. 11). Only the genomes of non-fern plants so far examined). (iui) Some one of the four fully characterized genes encodes a poten- defective CAB genes have been specifically amplified in the tially functional protein. Additional CAB genes isolated also genome of P. munitum. appear to be defective. The results are discussed in view of CAB gene family evolution and the implications for the The average haploid chromosome number for homosporous evolution of the fern genome.§ ferns, 57 (1), is so high that up to 95% ofall species have been considered polyploids (2, 3). However, it has been shown (4, 5) that within fern genera, species having the lowest chro- MATERIALS AND METHODS mosome numbers (n = 27-52) have the number of isozymes Plant Material. The genomic library was constructed from typical of diploid seed plants (6). These original observations a single plant of P. munitum (Kaulf.) Presl collected from a have now been expanded to include all the major groups of natural population near Saint Maries, Idaho (collection des- homosporous pteridophytes (reviewed in ref. 7). Therefore, ignation, Soltis and Soltis these plants either have gone through repeated cycles of 1845). This plant was subsequently polyploidy and gene silencing or were initiated with high maintained in greenhouse culture at Washington State Uni- chromosome number. If these species are ancient polyploids versity. that have "diploidized" by the accumulation of mutations in Construction and Screening of P. munitum Genomic Li- the extra copies ofgenes so that only one homeologous locus brary. Nuclei isolated by a sucrose gradient procedure (12) is now expressed, one would expect to find in these fern were then treated with hot cetyltrimethylammonium bromide genomes the remnants of the mutated and silenced genes. and the DNA was further purified (13). Construction of the The chlorophyll pigment molecules, the primary receptors library followed Maniatis et al. (14). The P. munitum DNA of light energy in the two photosystems (PSI and PSII) of (8.5 ug) was partially digested with the restriction endonu- eukaryotic photosynthetic organisms, are anchored in the clease Sau3A1. After size fractionation by sucrose gradient thylakoid membranes of the chloroplast through their inter- centrifugation, fragments in the 15- to 20-kilobase range were action with a set of proteins known as the chlorophyll ligated to the arms ofthe phage vector Charon 35. The library a/b-binding proteins (CAB proteins). In many angiosperms, was screened with probes derived from tomato CAB genes. both dicots and monocots, the CAB proteins are encoded by Specific probes are described in Results. Screening at low- a set of structurally and evolutionarily related nuclear genes, stringency conditions was carried out as described (9). i.e., by a "gene family" (8). So far, genes encoding six types Isolation and Characterization of Clones. DNA fragments of CAB protein have been identified in angiosperm species from recombinant phages containing P. munitum DNA that (see relevant citations in ref. 8). Sequence comparisons Abbreviations: CAB protein, chlorophyll a/b-binding protein; PSI and PSII, photosystems I and 11. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M29584). 195 Downloaded by guest on October 2, 2021 196 Botany: Pichersky et al. Proc. NatI. Acad. Sci. USA 87 (1990) hybridized to tomato CAB probes were further cloned into probes derived from different parts of the tomato Cab-3C the plasmid pBluescript (Stratagene) for nucleotide sequence gene. The original probe used in the isolation of these clones determination. Sequencing was done by both the chemical hybridized to all six clones, as expected. However, a probe (15) and the enzymatic (16) method. Most of the sequences, derived from the 5' half of the gene, (from a HindIII site 5' including all regions where deletions and insertions were to the initiating ATG codon to the HindIII site in the middle found, were determined on both strands. of the gene; ref. 9) hybridized only to clones 3, 4, and 7. We further characterized, by nucleotide sequence determi- RESULTS nation, two ofthe clones (nos. 3 and 7) that hybridized to both We screened 3 x 105 phages from the primary P. munitum the 5' and the 3' CAB probes and two ofthe clones (nos. 6 and genomic library (unamplified) with a probe derived from the 8) that hybridized to only the 3' probe. The nucleotide se- coding region of the tomato PSII type I CAB gene Cab-3C quences of these four clones are presented in Fig. 1. Only (from the HindIII site in the middle of the gene to the Xba I clone 3 contains a potentially functional CAB gene. Sequence site 3' to the termination codon; ref. 9). This probe identified comparisons indicate that this gene contains an intron with more than 50 positive phages. Six of these, numbered 3 consensus splice sites (5'-GT ... AG-3') in the same position through 8, were plaque-purified. Restriction mapping of the as do PSII type II CAB genes of angiosperms (18, 19) and an six clones revealed all six to contain different segments ofthe unspecified CAB gene from a moss (20), and it encodes a P. munitum genome (data not shown). We used DNA isolated protein of 265 amino acids. The assignment of the intron was from these phages in Southern blot (17) experiments with facilitated by the relatively high conservation ofthe amino acid A B roc 60 CCCT 6o :ft I120 mca 120 CA I150 160 ;AT;240 240 uc 300 PlTMSAT 300 :CT 1360 __c 360 8S T . K A L T R C SP 8 S e A Q A L L S S 420 ACAGACAAGCTGATGAGTAGI ~S~AAGCC0ICTCACeSGAGCOGACTACTCTC 420 8 T S Q Q L P V N Z L 8 R XK V A Y S T R L N 3 V A C X a G L D G A D T 8 GTGAGGCCC T (AAGTGCCCAACAGCCCCGCGATCICG 4650 GCATC&CCATGGTGGCCAI ICACACTCATCTGAAACCPA^CATCATA 450 G Z A R V Q N N A P I s x A P s c S I V R I T N I R A A T P N S I V AACTIOCCCCCGAGETAGACATTC¢CCTCATGATCC_ _ !GA S40 AGIT~TFVGATGflCATGCACCAG;CATTGATITSCTTCATFCIY CTGCATVAT 540 600 -1 660 TGATCATG~>AcGGCCCRAC~4A_rGPCTGGGCOCC1YCAGTGGGTC 600 720 I G P_ N_ K (P) K F L G P FS C I S 760 CCAT~TCC¢S GCGGACI0GCACS;SCTGACAA C LGWIC 660 _GITCAIAATOOCVOOTCSG=OOCMCGAM GCCCTCTACTTOCCC 840 P 8 I L KG FE Y 8 8 D Y C V D I A G L 8 Y G S D R P L Y L G P 720 900 A D P X T F A N R E L E V I EV8ARa TCGCGCTCTTF S C __W8 P P 8 Y C10FOL 8 G GCF 7 CP GGOACTAT(D Y G VCCGD CTC N OlCGTOAAA G OAaCOCGOOTACTAOCAAAO GGTCCAST 760 960 L L 0 T L E I V T P Z I L A KN G V P I T A G L 8 A D P E T F A X N RE L E Y I AAGA CCAOATOTCAAOOAAIA(ALTCAGlAG0 LACTA 540 ACTCTAGATOOCAAGCCWIICSCIM OTTTA C J CCGGAlCTC'IOCi/ A 1L020 K E PV VPF I A Z A Q I FS EGG L D Y E S R V A M L GA L G C V T P Z L L A X 5TIOC0ACC:ACGTIO&CATCACASGJ TM _GCAOTI CCCI~AGT too 1080 L C N P A L I R AQ I L A T L A K Q V N G V G E A V V F x A G S Q I P A E OTOCTLfGACATGAAGCGTAAGC OAEIGCCAT C~AGTGATAI S 960 r r I E I OCOCClO4 ATATOOC JCcXCCAcTGTCAO CATA1 1IN¶ CATCT 11140 V L L O a X A Y it V A R P L G X V 0 G L D Y L G N P S L V M A Q S I L A I OATCCAOTTCcOGcGGTGCTI7 OCSCCCAITLCICOACiAOCAGAT C 1020 OGCC70 =~TOICATCTIGGOTTGAOA !~~CT( ICN LOCAC 11200 D P V Y P G G A P D P L R L A D N P D A V A C Q V I L N G A V E G Y R V A G G P TTGOCAGrAAGGTFAAAGAGITAAATC0AOCGOCAT@ ANI rOCTIS:1080 TarOOTOAG rz IAOcOACCCC CTACCCIN ta~OGG~] C LIIcccc c 11260 L a I L K V K E I K N G R L A K F V A F L G r v E D P I Y P G G SF D P L G L A OOA S~lCCTCOTOJC GGCAA-- CCCATCTAGAAC :i ITACCC3C1140 ATGACCC CGAACCTTCCA AGAAGACTGA AATGGTGGCA 11320 C F F C Q- A I V -TG I G P I * N L D R D D P E A F A Z L K V I Z L K N G. RL A CTCGCCGACCT A C ACTC XTAA 1200 I TCT GCCAWGCCAGTCT ACTGTAAAAGGTCCCATTGAGA I1380 L A D P V A N N A N A IY T S F V P G A xN s N F. 0 F F V Q A I V T G K G P I E TTCGATCAASCTAOCCCSATAAAAAGCCCTAACTOATAC AAA 1260 ACITCTG CT( 1 TCACCTTGCTOACCCCOACAOTTAATACC GCCI ~GCC~T 'A GCCACTAACT I1440 ATTCGTCTCA!GCCIGF D L Q C Y a L N L S D H L A D P A V N N AW A Y A T N 1260 TCACCCCCGGCAAGTGAGAAAITCITI'TTTCTTCTTACTGCATTCGGGTAGCCGAAGCGTT I1500 F T P G X D CGCCCCCSTr CTACACJTACATCTAT ATTAGCTTATCTTCTACTGTTGTCAT I1560 CCTCTrTSG^TTSGTACTSrCTCAACTCCrTTCCTTAACTAATCTTTTATGTGAI1620 60 AA^TYATTATGTGGCCTCG~cTTMTTAOTTOTGGATATGTCGCIT1TTTTT¶TACCTCG I1680 -6 SCACAAX.TG~ATACAOAOTGAAGAOTCATOTTOCAATA GTAACGTAAAAAAAATGTTT I 1740 GCCTCTGCTCAAGG CAACTG 120 TTCTACCAACGAAAATAAC AATTTCTSAGCCCGGTCTTGCTTGAGAAATTTAGTAA I1800 H A * S I L A I N Q V V L X 1846 CCCAAGTAAATGGCAAAATCAACCATCl TCAAGGCTAGC G«GAG=GCTACCCCATPAATWACTGGTA _A_ A _TTC__ 180 F G Y P I N G L A G V G E G G D L Y L G -1 -24 C AGGCCAGTACTrT"SCAc CCGCCCGCAC~AGOcCAATGAA240 0 Q Y F (?) P L C L T D D P K G L K N G R TTITEAAATTGAATAAAAACCCTCCCATTAAI?1!AATCCTTAAATTATCTCAAAAAA 60 CIOCTATGTTCTTIIICGATTATC;AACTACATCT ;u GC 300 TATAATTAAAAACCCC CA UGCA 120 L A N F S N F G F F V Q A I I D T S 1 G -S CCTCOAAAA1¶¶GCTAOACCACCTOGATA~cC~?1GCAAACAATTOCTT'GCCTAT 360 Cccc _ _ _GCTTCCAAGT_ 10 P L E N L L D H L D N P V A N N A V A Y Q V V L E G Y P I N E L A S GCTACCAAAG1GTCCCCGTGCTT^ GTACATCCaTCCCCCATCACTAOTC 420 GTGGGGAGGGGGGACATCTATACCTTAGAGGCCAATACTCTGACCCCCTCGGCCTCGCA 240 A V G E GG D. L Y L R G Q Y F D P L G L A 460 GACGACCCCAGCACCT TGCAAGACTTGTT 300 540 D D P S T L. A X L X V X E L K N G R L V 600 ATGTTCTCCATC=:. CATSATTATCACTAOAAAAGOCCCACTTAA 360 660 N F s X F G L F V E A I I T R X G P L E 720 AAMTTGCTACACCACCTSGATAACCTATIGCAAACAAOGTTAACCAGGTOAGTAACCCT 420 780 N LLD L D N P I A N 40 CTrGGGTTTCACCTTAAAAAAATTTAGTOTACCACOTOCAGTTCAAATCaATOOOTATT 430 900 960 TTAATTAGATAAATTGAAAA 500 1020 FIG. 1. Nucleotide sequences of fern CAB genes and flanking regions. (A) Sequence of the gene on clone 3. The amino acid sequence of the encoded polypeptide is shown (in one-letter code) below the DNA sequence. Underlined are the consensus 5' and 3' splice sites of the intron (GT ... AG). (B-D) Sequence ofthe CAB gene on clones 7, 8, and 6, respectively. Positive numbers indicate sizes of insertions (in nucleotides) and negative numbers indicate sizes of deletions (all insertions and deletions are relative to both the fern CAB gene on clone 3 and the tomato CAB gene Cab-3C; in regions where Cab-3C and the fern clone 3 are not colinear, no insertions/deletions were inferred). Asterisks indicate stop codons in the middle of the coding region or directly downstream from it. Only the amino acid residues specified by the CAB-related sequences are shown. In C, arrows underscore positions of the inverted repeats flanking the sequence with homology to CAB sequences. Downloaded by guest on October 2, 2021 Botany: Pichersky et al. Proc. Natl. Acad. Sci. USA 87 (1990) 197

30 60

T3C: . M.S.STFAGKAVKL. SPS.SEIT !NG. .T.RKTAT..KPAS...P...... T...... F3: MATSTAALTSTFSGQQLKPV NELSRKVG AGEARVQMMAPKSKAP SGSIWYGSDRPLYLGPFSGSPPSYLSGEFPGDYGWDTAGLSAD F7: . .LTRCSFS.CAQALLSSYSTRL. .VAC.A.LDG.DYS.IT.RR---A.T PN...PN.-KF .. ..ES.....K. .YSS ... I.... 90 120 150 180

T3C: . C.F. R. S . V.I. F3: PETFAKNRELEVIHSRWAML-GALGCVTPELLAKNGVKFGEAVWFKAGSQIFAEGGLDYLGNPSLVHAQSILAIWACQVILMGAVEGYRVAGGPLGE F7: K ...... P...... T.L...... TL...... MKA.....VRE... F6: [...--..V..---...PIN.LAGVG F8: (..V. .---..*.PINELASVG 210 240 265

T3C:.V..L ...... I...... A. VN .F.... V... F3: VEDPIYPGGS FDPLGLADDPEAFAELKVKELKXNGRLAMFSMFGFFVQAIV TGKGPIENLSDHLADPAVNNAWAYATNFTPGK F7: .I. ....VA .. R . N.D.L.K. I . VA ...... *. VA. T.S.V ..AFDLQGYAL F6: EGGDL.L ..QY.-..... ------. G..... ID.S .L L.. .L.. DN.VA. ] F8: EGGDL.LR.QY.STL.K. V .L..H..I P.R.. L..L...DN.IA.] FIG. 2. Sequence comparisons of the protein-coding information of the fern CAB genes on clones 3 (F3), 7 (F7), 6 (F6), and 8 (F8) and the tomato gene Cab-3C (T3C). Only the F3 sequence is shown in full. Identical residues in the other proteins are indicated with dots. Brackets in the F6 and F8 sequences indicate the extent of the coding information found in these sequences. A dash represents a deletion in the defective gene(s) relative to F3 and T3C. Some gaps in the sequences have been introduced to maximize similarity without inferring direction of change (insertion or deletion). sequence encoded by the parts of exon 1 and exon 2 flanking produce any additional signals, indicating that the missing the intron. The part ofthe protein encoded by the first exon of parts of these genes do not reside in the immediate vicinity the gene from clone 3 consists ofthe 36-residue transit peptide (-5 kb upstream and downstream; data not shown). How- and the first 13 residues ofthe mature CAB protein. The amino ever, sequence determination of clones 6 and 8 did reveal a acid sequence encoded by the first exon (with the exception of shared sequence, unrelated to CAB sequences, in the region the first few codons and the last five codons) is highly variable upstream to each CAB-related sequence (data not shown). even within angiosperms, and the fern sequence of this exon is also highly diverged from other known CAB sequences. The DISCUSSION second exon ofthe fern CAB gene on clone 3 encodes a protein The CAB gene from clone 3 has an intact structure typical of with >85% sequence identity to other known PSII type I and other known functional CAB genes from moss (20) and type II CAB proteins (Fig. 2, Table 1). This CAB gene does not angiosperms (8). The protein it encodes shows high sequence contain any obvious structural defect (but see Discussion); similarity to the previously reported moss CAB sequence and however, we have not determined whether it is transcribed to PSII type I and II CAB sequences of angiosperms (Table and, if it is transcribed, whether a functional protein is pro- 1). However, it does not contain the highly conserved se- duced and is properly transported to and assembled in the quence Met-Arg-Arg or Met-Arg-Lys at the processing site of chloroplast. This gene can thus be considered only as a the transit peptide but instead contains the sequence Met- "potentially functional" gene. Met-Ala (Fig. 2). Thus, although a protein with high sequence The CAB genes from the other three clones were revealed identity to other known CAB proteins may result from the by nucleotide sequence determination to be clearly defective. expression of this gene, further work is necessary to show The CAB gene on clone 7 contains both exons and the single whether indeed this intron in the same position as that in the CAB gene on clone gene is transcribed and, if so, whether a 3. However, in the second exon it contains a deletion of a functional protein is produced and is properly transported to single nucleotide, as well as two nucleotide substitutions that and assembled in the chloroplast. result in nonsense (termination) codons (Figs. 1 and 2). The In angiosperms, the PSII type I genes contain no introns (9) overall sequence identity of the coding region of this gene to but the type II genes do contain a single intron (18, 19). This the CAB gene on clone 3 and to angiosperm PSIICAB genes intron is present in the moss CAB gene and in the two fern is much lower than the sequence identity of clone 3 to the sequences described here that contain sequences homolo- angiosperm CAB sequences. The CAB genes on clones 6 and gous to both the 5' and the 3' coding region of CAB genes 8 each contain the central portion of the second exon only, (i.e., the CAB genes from clones 3 and 7). Thus, a CAB gene with the first exon completely missing. Within the segment of containing no introns has not yet been identified in any homology, the sequences from clones 6 and 8 show additional species other than angiosperms. It is likely that the PSII type deletions and insertions relative to known functional CAB I CAB genes of angiosperms represent a lineage that has lost genes. In addition, they also contain nonsense mutations this intron. The angiosperm type I and type II CAB genes (Figs. 1 and 2). Probing these clones with short probes from (represented by tomato) encode proteins that are only 12% the extreme 5' and 3' coding regions ofthe CAB genes did not divergent from each other in the region compared in Table 1. In the same region, the protein encoded by the fern CAB gene Table 1. Sequence identity (percent) of the protein segment from clone 3 is slightly more similar to the tomato PSII type encoded by the second exon of CAB genes of fern, moss, I protein (9.7% divergence) than to the tomato PSII type II and tomato protein (13% divergence), suggesting that the angiosperm F3 Ml I-T3C PSII type I and type II CAB gene lineages diverged slightly before, or perhaps immediately after, the separation of the Ml 87.0 fern and angiosperm lineages. Other types of angiosperm I-T3C 90.3 87.5 CAB genes encode proteins that show much greater diver- I1-T5 87.0 84.3 88.0 gence (60-70%o) (1), and the splits among these lineages are F3, fern CAB gene on clone 3; Ml, CAB gene of a moss (20); therefore likely to have occurred prior to the divergence of I-T3C, tomato PSII type I CAB gene Cab-3C (9); II-T5, tomato PSII lineages leading to angiosperms and ferns. Genes for these type II CAB gene Cab-5 (21). additional CAB lineages have not been isolated from ferns. Downloaded by guest on October 2, 2021 198 Botany: Pichersky et al. Proc. Natl. Acad. Sci. USA 87 (1990)

The sequences found on clones 6, 7, and 8 represent cally studied on the molecular level, but some angiosperm CAB-related sequences. However, structural defects (inser- plants of known recent polyploid origin, such as hexaploid tions, deletions, nonsense mutations) clearly render these wheat (Triticum aestivum, n = 21), have been shown to CAB genes nonfunctional. In each case, the portion of the maintain multiple functional copies of genes encoding many, gene that has survived also shows a high rate of coding but not all, isozymes (25). P. munitum and other ferns with sequence divergence compared with the known functional similar chromosome numbers (n = 27-52) do not show CAB genes (Fig. 2). The large proportion of nonfunctional multiple copies of functional genes encoding most isozymes PSII CAB genes in P. munitum is in marked contrast to the (4, 5). If these ferns are in fact descended from polyploid situation in angiosperms, where a detailed investigation of lineages, as suggested by their chromosome number, it must several species has revealed few or no clearly defective CAB be assumed that the polyploidization events in the history of genes. For example, the tomato genome contains eight func- these lineages are much more ancient than the polyploidiza- tional type I and two functional type II CAB genes and only tion event that gave rise to wheat. Alternatively, gene silenc- a single truncated type I gene (9). ing in fern polyploids may occur much faster than in an- A possible trivial explanation for these observations is that giosperm polyploids. artifacts in the cloning procedures have produced DNA For several reasons, some of which depend on the specific sequences that do not reflect the situation in vivo in the P. gene system examined (CAB) and some of which do not, it is munitum genome. Two observations argue against this pos- not possible with the present data to distinguish among the sibility. First, artifacts in cloning usually involve large dele- above. In the P. tions and insertions (22), but not short deletions and inser- three competing hypotheses listed screening these changes have munitum DNA library, we identified more than 50 positive tions and nucleotide substitutions, and all and the first 6 we further characterized were all in genes. observation that phages, that been found the cloned Another different, suggesting that the number of CAB genes in P. suggests that the cloned sequences are accurate representa- ifthe tions of sequences in the genome is the similarity between the munitum is considerably greater than 6. However, even defective CAB genes from clones 6 and 8. For example, both number of CAB genes in P. munitum were much higher than contain an eight-nucleotide deletion in the exact same posi- that encountered in diploid angiosperms (at present, the tion (Fig. 1), and the sequences of the encoded proteins are known range is 3-17; refs. 19, 26, and 27), it would still not very similar to each other but very different from other be possible to distinguish between specific gene amplification known CAB sequences (Fig. 2). Clones 6 and 8 are indepen- and ancient polyploidy. On the other hand, because PSII dent isolates from the primary library construction (unam- CAB gene number varies widely even within diploid an- plified) and it is, therefore, very unlikely that the many small giosperms, a relatively low copy number (6 or a few more) changes that are shared by both sequences have occurred cannot rule out the ancient polyploidy model either. Although independently during the construction and handling of the discriminating among the hypotheses we present here is library. further complicated by the fact that we have examined a gene Several scenarios could be invoked to explain the existence family with a variable number ofgenes in different organisms, of multiple defective CAB genes in the genome of P. muni- in principle the same difficulties will also arise when single- tum. First, some defective CAB genes may have been copy gene systems are examined. This is so because if specifically amplified, by unspecified mechanisms, in the multiple copies (defective or functional) are found, ad hoc genome of P. munitum during evolution. Unusually large explanations can again be invoked-for example, specific numbers of copies of a specific gene in some species have gene amplification. been observed previously. For example, the genome of Thus, in practice, gene silencing and, by implication, the petunia contains >200 copies of actin genes, whereas other ancient polyploid origin of a fern species with n = 27-52 (or plants examined have at most 10-15 copies (23). It is not of any other polyploid) can be demonstrated only when the known, however, whether any of the petunia actin genes are presence of multiple defective genes is shown to be the case defective. In mammalian genomes, multiple nonfunctional notjust for one or a few gene systems (each system may have "pseudogenes" are often created through reverse transcrip- its own peculiarities) but for most nuclear gene systems, tion of mRNA and integration of the copy DNA into the including gene families and the genes usually found in single chromosome (24). The defective CAB genes whose se- copy in diploid seed plants. The accumulation of these kinds quences we present here are unlikely to have been amplified of data is therefore of the utmost importance in the study of by an RNA intermediate, since one of them (on clone 7) genome evolution in general and in particular in polyploid contains an intron, and the other two (on clones 6 and 8) are plants, both in ferns and in other types of plant groups, and missing both 5' and 3' ends of the coding region and show no the molecular techniques (e.g., gene cloning and sequencing) evidence of a poly(A) tail (Fig. 1). The similarity between to carry out such investigations are now available. clones 6 and 8 is intriguing. They may indeed represent an Although it is thus premature to conclude that our results amplified sequence in the genome, or they may simply be indicate an ancient polyploid origin for P. munitum, it is allelic (with extensive deletions on both homologous chro- intriguing that this study, which to our knowledge is the first mosomes at this locus). molecular examination of a fern nuclear gene system, de- A second scenario assumes that the P. munitum lineage has tected multiple defective genes. If defective copies are found never undergone a polyploidization event, and that starting in most other gene systems in ferns, it would be of great with a normal complement of PSI1 CAB genes, several, and interest to examine the effect such nonfunctional genes have perhaps the majority, of the members of the PSII CAB gene on the expression of the fern genome, and, in consequence, family in the genome of this plant have mutated to a non- of the functional state. As discussed in the Introduction, this phe- on the cellular and developmental processes plant. in nomenon has not been observed in the CAB gene family We thank B. E. Froman for assistance and advice in the construc- other plant genomes or in other angiosperm gene families. tion of the genomic library, Dr. L. D. Gottlieb for providing his lab A third scenario is based on the assumption that P. facilities and advice in the library construction, D. Stein for providing munitum was, sometime during its evolution, a polyploid. us with some DNA samples, Dr. A. Zilberstein for disclosing her Then its present genome may have undergone the process of unpublished results (which may be similar to ours) and Dr. L. D. diploidization, which, among other things, also involved Gottlieb, C. H. Haufler, and W. H. Wagner for critical review of the mutations to nonfunctionality in extra copies of genes (4, 5). manuscript. This work was supported by U.S. Department of Gene silencing in plant polyploids has not been systemati- Agriculture Competitive Research Grant Office Grant 88-37262-3878 Downloaded by guest on October 2, 2021 Botany: Pichersky et al. Proc. Natl. Acad. Sci. USA 87 (1990) 199 (to E.P.) and National Science Foundation Grants BSR 8620444 (to 13. Saghai-Maroof, M. A., Soliman, K. M., Jorgensen, R. A. & P.S.) and BSR 8607054 (to L. D. Gottlieb). Allard, R. W. (1984) Proc. NatI. Acad. Sci. USA 81, 8014- 8018. 1. Klekowski, E. J. & Baker, H. G. (1966) Science 153, 305-307. 14. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular 2. Grant, V. (1981) Plant Speciation (Columbia Univ. Press, New Cloning (Cold Spring Harbor Lab., Cold Spring Harbor, NY). York). 15. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 3. Wagner, W. H. & Wagner, F. S. (1980) in Polyploidy: Biolog- 499-560. ical Relevance, ed. Lewis, W. H. (Plenum, New York), pp. 16. Sanger, F., Nicklen, S. & Coulsen, A. 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