Genes Encode Highly Similar Chloroplastic NADP-Malic ...that Mel expression in leaves parallels the degree of C, photosyn- thesis in seven Haveria species. Furthermore, whereas Me2

Plant Physiol. (1996) 1 1 1 : 1251-1261

Two Genes Encode Highly Similar Chloroplastic NADP-Malic Enzymes in Haveria

lmplications for the Evolution of C, Photosynthesis

Jerry S. Marshall’, John D. Stubbs, and William C. Taylor*

Commonwealth Scientific and Industrial Research Organization Division of Plant Industry, G.P.O. Box 1 600, Canberra, Australian Capital Territory 2601, Australia (J.S.M., W.C.T.); and Department of Biology,

San Francisco State University, San Francisco, California 941 32 (J.D.S.)

To gain an understanding of the molecular events underlying the evolution of C, photosynthesis, we have undertaken a detailed study of the NADP-malic enzyme gene family in C, and C, species of Haveria. Three genomic clones from the C, species Haveria bidentis were characterized and found to encode two highly similar chloroplastic forms of NADP-malic enzyme, termed ME1 and ME2. Genomic Southern blotting with gene-specific probes showed that both Me7 and Me2 are found in Haveria trinervia (C,) and fiaveria pringlei (C,) as well as in F. bidentis. Northern blots demonstrated that M e l expression in leaves parallels the degree of C, photosynthesis in seven Haveria species. Furthermore, whereas Me2 was expressed at a low level in both roots and leaves of f. bidentis, M e l expression was seen only in leaves and was light-regulated. We discuss these results in the context of the evolution of C, photosynthesis in Flaveria.

The C, photosynthetic pathway is a recently evolved modification of the C, pathway in which plants spatially separate the initial fixation and subsequent conversion of CO, to 3-phosphoglycerate and other carbohydrates. Through the use of this system, C, plants effectively concentrate CO, in the vicinity of Rubisco (located in bundle-sheath cells) and, thus, avoid the wasteful process of photorespiration. The efficient operation of C, photosynthesis requires the strict compartmentation of a suite of enzymes in either mesophyll or bundle-sheath cells. This intercellular compartmentation is a distinguishing feature of C, plants: a11 of the key enzymes utilized in C, photosynthesis are present in C, plants; however, their expression patterns are distinct from those seen in C, plants. It is generally accepted that C, plants evolved from C, ancestors and that the specific enzymes utilized in C, photosynthesis derived from corresponding enzymes present in the C, ancestors (Cock- burn, 1983). In the evolution of C, photosynthesis, the expression programs of these ancestral proteins were

Present address: Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, Canberra ACT 0200, Australia.

* Corresponding author; e-mail [email protected]; fax 61-6- 246-5000.

I251

modified to provide the level and cell specificity required for efficient functioning of the C0,-concentrating mechanism.

The nature of the modifications required to redirect an enzyme for use in C, photosynthesis depends on the enzyme involved. Relatively small changes would be required to confer bundle-sheath-specific expression on Rubisco. Other aspects of its expression program (e.g. light regulation, organ specificity, and intracellular location) need not have changed dramatically. However, the changes required for other enzymes were likely much greater and may have involved duplication of the ancestral gene to provide a copy that could be manipulated in evolution without having deleterious effects on critica1 func- tion(s) performed by the ancestral enzyme. Thus, an understanding of the evolution of C, photosynthesis requires knowledge of changes in both gene copy number and expression between C, and C, species.

A number of recent studies have utilized the genus Flaveria to analyze the molecular events underlying the evolution of C, photosynthesis. This dicotyledonous genus is particularly well suited for such studies because, among its 21 species, several utilize C, photosynthesis, several others utilize C, photosynthesis, and a number of species are anatomically and physiologically intermediate between C, and C, (Raws- thome, 1992). It is therefore possible to study the molecular differences between closely related C, and C, Flaveria spp. without the added complication of evolutionary changes that have accumulated during prolonged separation of less closely related species.

Severa1 key enzymes involved in C, photosynthesis in Fla- veria have been studied at the molecular level. Perhaps the best characterized of these is PEPCase, wluch is encoded by a complex multigene family. The C, isozyme of PEPCase is encoded by several genes (Poetsch et al., 1991) making up a single subfamily, ppcA (Hermans and Westhoff, 1990, 1992). The ppcA subfamily is present in C, as well as C, species of Flaveria. However, the characteristic high-level, leaf-specific expression pattern is not seen in C, species. Thus, for PEP- Case, it would appear that gene duplication events occurred

Abbreviations: ME, malic enzyme; PEPCase, PEP carboxylase; 3’ UTR, 3’ untranslated region.

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1252 Marshall et al. Plant Physiol. Vol. 1 1 1, 1996

prior to the separation of C, and C, species, leading to a nove1 ppc subfamily that was subsequently modified to provide the expression pattern required for C, photosynthesis. In contrast, two other Flaveria C, enzymes appear to be encoded by single genes: the mesophyll cell-specific enzymes pyruvate, Pi dikinase (Rosche and Westhoff, 1990) and NADP-malate dehydrogenase (McGonigle and Nelson, 1995).

A fourth Flaveria C, enzyme that has been analyzed at a molecular leve1 is chloroplastic. NADP-ME, which is located in bundle-sheath cells. Full-length cDNA clones encoding NADP-ME have been reported from the C, species Flaveria trinervia (Borsch and Westhoff, 1990) and the C, species Flaveria pringlei (Lipka et al., 1994). In addition, a partial cDNA clone has been reported from the C,-C, intermediate species Flaveria linearis (Rajeevan et al., 1991). Based on South- ern blot analysis using partial NADP-ME cDNAs from F. linearis and F. trinervia to probe genomic DNA from these two species and from F. pringlei, Rajeevan et al. (1991) concluded that NADP-ME is encoded by a small gene family, possibly consisting of three members. The nature of these genes and the number that are involved in C, photosynthesis were not determined. However, it seems likely that at least one of the Flaveria NADP-ME genes encodes a cytosolic enzyme, since cytosolic NADP-MEs have been identified in a number of diverse plant species (Walter et al., 1988; Van Doorsselaere et al., 1991; Cushman, 1992; Franke and Adams, 1995). The proteins encoded by the F. trinervia and F. pringlei NADP-ME cDNAs are highly similar and contain apparent chloroplast transit peptides of identical length. In addition, Southem blotting under conditions designed to exclude detection of distantly related genes indicates that both cDNAs derive from single genes. Based on these data, Lipka et al. (1994) concluded that the F. trinervia and F. pringlei cDNAs represent orthologous genes and, therefore, the C, NADP-ME isoform in F. trinervia evolved from a chloroplastic NADP-ME already present in C, ancestral species.

Our primary interest was in elucidating further the molecular events that occur in the evolution of the C, isoform of NADP-ME in Flaveria, particularly the modifications that give rise to the high-level, bundle-sheath-specific expression of this enzyme. To analyze the promoter regions of the gene encoding the C, NADP-ME, we began by isolating a number of NADP-ME genomic clones from the C, species F. bidentis. In the process of characterizing these clones to determine which encoded the C, isoform, we discovered that the NADP-ME gene family in Flaveria is more complex than previously thought and that chloroplastic NADP-ME is encoded by two distinct and yet highly similar genes in F. bidentis, F . trinervia, and F. pringlei. We have used gene- specific probes to analyze the expression of both.genes and have determined that only one of the genes is likely to be involved in C, photosynthesis in a range of Flaveria spp. In this paper, we discuss the implications of these findings in the context of the evolution of C, photosynthesis in Flaveria.

MATERIALS A N D METHODS

Plant Material

Flaveria bidentis and Flaveria pringlei seeds were obtained from Dr. M.S.B. Ku (Washington State University, l‘ull-

man). Flaveria palmeri, Flaveria brownii, Flaveria linearis, Fla- veria trinervia, and Flaveria floridana seeds were obtained from Dr. R.H. Brown (University of Georgia, Athens). Plants were maintained in a greenhouse in Canberra, Australia.

lsolation of c D N A and Genomic Clones

A partial cDNA encoding maize chloroplastic NADP-ME (a generous gift from Tim Nelson, Yale University, New Haven, CT) was used to screen a AgtlO cDNA library prepared from F. bidentis leaf mRNA. A single clone, termed Ame1 1, was selected and plaque-purified. The 0.5-kb insert of this clone was sequenced and its identity confirmed by comparison with the published maize and F . trinervia NADP-ME cDNA sequences (Rothermel and Nel- son, 1989; Borsch and Westhoff, 1990).

The insert of hmell was used to screen a F. bidentis genomic DNA library prepared using DNA partially digested with Sau3Al and ligated into AGemll (Promega), according to the supplier’s instructions. Seven positive clones were purified by successive rounds of screening. Recombinant phage DNA was prepared, and clones containing the 5‘ end of the NADP-ME gene were identified by Southem blotting using the oligonucleotide MEMAT (see below), end-labeled with polynucleotide kinase as a probe (Sambrook et al., 1989). DNA was transferred to Nytran membranes (Schleicher & Schuell), and blots were hybridized in 6X SSC, 5X Denhardt’s solution, 0.5% (w / v) SDS, and 50 pg / mL salmon sperm DNA at 52°C and washed 5 min in 6X SSC, followed by 5 min in 2X SSC, both at 52°C.

Oligonucleotides

The NADP-ME oligonucleotides used were: MEMAT,

universal 5’, 5’ -TTTGCAAACCACAACGCC; NADP - universal 3’,5‘-CTCCAGAGCTATGAGTTC; ME interna1 5’,5‘-GTCACAGACGGTGAACGAATCTTAGG; ME intemal 3’,5’-GCTGTGTACAGAGCAAGCTITCCCACAGG; ME term 5‘,5’-CTCCAGCTGAACAGGTGACACAGG; ME term 3’,5’- CGGTAGATGCGGTATITGGGGCTG; ME 3’ universal, 5’- GAGAG(C/T)TGCATGTACAG(C/ T)CC; Mel 3‘ LJTR 5’,5’- CCCGAAlTCGTITAGCGGGGGAAAAAGGACAG; Mel 3‘ UTR 3’,5’-CTATAAACGGCCAGCTTITAG-CTAC; Me2 3’ LJTR 5’,5’-CTITACGGTGAATCTGATAC; Me2 3’ UTR 3’,5’- CTAATGACTGACAACTAAG.

5‘ - TGGCGGTATCCTCACCGTACATATCCTC; NADP -

D N A Sequencing

DNA sequencing was accomplished using an Applied Biosystems model 373A DNA sequencer with either dye primers or dye terminators. The 5’ and 3’ regions of M e l and Me2 were sequenced in both directions.

Cenomic D N A lsolation and Southern Blot Analysis

Leaves (2 g fresh weight) were ground in liquid nitrogen, and the frozen powder was suspended in 5 mL of extrac- tion buffer (100 mM Tris-HC1, 100 mM NaCl, 50 mM EDTA, 2% (w/v) SDS, 2% (v /v) 2-mercaptoethanol, and 100



Two Genes for Chloroplastic NADP-Malic Enzyme in Haveria 1253

pg/mL proteinase K, pH 8.5). After the sample was incubated 1 to 2 h at room temperature, 10 mL of Tris-EDTA (50 miv Tris and 1 mM EDTA, pH 8.0) were added, and the solution was extracted three times with an equal volume of pheno1:chloroform (l:l, v / v). Nucleic acids were precipitated by the addition of 0.1 volume of 3 M sodium acetate (pH 6.0) and 1 volume of ethanol. The resulting pellet was dissolved in Tris-EDTA, RNase A was added to 100 p g / mL, and then the sample was incubated for 1 h at 37°C. The solution was extracted with pheno1:chloroform (l:l, v/ v), and genomic DNA was precipitated by the addition of 0.1 volume of 3 M sodium acetate (pH 6.0) plus 2 volumes of ethanol.

For Southern blots, 10 to 15 pg of genomic DNA was digested with appropriate restriction enzymes, and the resulting fragments were separated by electrophoresis in a 1% (w/v) agarose gel. DNA was transferred to a Hybond N+ membrane (Amersham) as described by Sambrook et al. (1989). Gel-purified PCR products were labeled with 32P by random oligonucleotide priming using an Amersham Megaprime kit. High-stringency hybridization was in 6X SSC, 0.5% (w/v) SDS, and 100 Fg/mL salmon sperm DNA at 65"C, followed by two washes of 15 min each in 2X SSC plus 0.2% (w/v) SDS (65°C) and two washes of 15 min each in 0.2X SSC plus 0.2% (w/v) SDS (65°C). Low-stringency hybridization was in 6X SSC, 30% (v/v) formamide, 0.5% (w/v) SDS, and 100 pg/mL salmon sperm DNA at 42"C, followed by washings as above except at 42°C.

RNA lsolation and Northern Blot Analysis

Total RNA was isolated from leaves and roots of greenhouse-grown plants following the REX RNA isolation protocol (United States Biochemical). Poly(A)+ RNA was isolated using a PolyATtract kit (Promega). Total RNA (10 p g ) or poly(A)+ RNA (from 500 F g of total RNA) was separated by formaldehyde agarose gel electrophoresis and transferred to nitrocellulose or Hybond N (Amersham) membranes as described by Sambrook et al. (1989). Blots were probed and washed under low- stringency conditions as described above.

For analysis of the light response of Mel expression, greenhouse-grown plants were transferred to total darkness, and leaves were harvested after 2 and 5 d. After 5 d in darkness, plants were placed in a light cabinet (PPFD of 800-900 pmol m-' s-') and leaves were harvested after 2, 4, 8, and 24 h of continuous illumination. Total RNA was isolated as above.

For use in normalizing RNA levels, a probe for actin was made by PCR. Two oligonucleotide primers were designed from conserved regions of six plant actin cDNAs: act5',5'- AC(A / T)GG(A/ T)ATGGTCAAGGC and act3',5'-GTGTG- GCTIACACCATC (where "I" denotes inosinic acid). F. bidentis leaf poly(A)+ RNA was reverse-transcribed using Superscript reverse transcriptase (BRL) according to the supplier's instructions, and the resulting cDNA was used as a template for PCR amplification. The resulting 446-bp PCR product was sequenced to verify its identity, radiolabeled, and used as a probe as described above.

Purification of NADP-ME from F. bidentis Leaves

NADP-ME from F . bidentis leaves was purified to elec- trophoretic and chromatographic homogeneity by sequen- tia1 fractionation with (NH,),SO, and PEG, followed by chromatography on DEAE-Sepharose as described below. The protein from the crude, soluble homogenate precipitating between 50 and 60% saturation (NH,),SO, was further fractionated with PEG 8000. The protein precipitating between 7.5 and 27.5% (w / v) PEG was adsorbed to DEAE- Sepharose and eluted with a KCl gradient. The major peak of NADP-ME activity emerging at about 200 mM KCI ex- actly co-eluted with a protein peak and had a specific activity of about 25 pmol min-l mg-' protein. SDS-PAGE analysis of these fractions showed a major band (mass approximately 63 kD) that made up about 95% of the protein on the gel. The remaining protein migrated as an approximately 60-kD species, which may be a proteolytically modified form of the NADP-ME polypeptide. After SDS-PAGE, the approximately 63-kD protein band was transferred to an Immobilon-P membrane (Millipore) and sequenced at the Biomolecular Resource Facility of the John Curtin School of Medica1 Research (Australian National University, Canberra). The details of the NADP-ME purification scheme will be published elsewhere (A.R. Ashton, unpublished data).

RESULTS

lsolation and Characterization of f. bidentis NADP-ME Cenomic Clones

To identify Flaveria NADP-ME genomic clones, we isolated a 0.5-kb partia1 cDNA from a leaf cDNA library from the C, species F. bidentis. This cDNA, termed hmell, was identified using a maize NADP-ME cDNA (Rothermel and Nelson, 1989) as probe. The nucleotide sequence of Ame11 was >99% identical with that of the middle region of two NADP-ME cDNAs previously reported to encode the C, form of the enzyme in the closely related species F . trinervia (data not shown; Borsch and Westhoff, 1990; Rajeevan et al., 1991).

The insert from hmell was used to probe an F. bidentis genomic DNA library, from which seven putative NADP-ME genomic clones were isolated. Based on similarities of restriction patterns, the clones were divided into three classes: Mel (two clones), Me2 (one clone), and Me3 (four clones). To screen for clones containing the 5' end of the NADP-ME gene, we used Southern hybridization with a radiolabeled oligonucleotide based on the region of the F . trinervia cDNA encoding the amino terminus of mature NADP-ME (termed MEMAT, Fig. 1C). This oligonucleotide hybridized to one clone in each of the classes Mel and Me2, but did not hybridize to any of the clones in Me3. We concluded that the clones in Me3 either did not contain the 5' end of the NADP-ME gene or were so dissimilar to the F . trinervia cDNA that they could not form a stable hybrid with the oligonucleotide. Because we were primarily interested in analyzing full-length clones similar to the F . trinervia cDNA (and therefore likely to be involved in C,



1254 Marshall et al. Plant Physiol. Vol. 11 1 , 1996

START

A Mel hmel STOP

I

hme7

START STOP

Me2 hme6

Figure 1. Restriction maps of F. bidentis NADP-ME genomic clones. A, Overlapping clones hmel (top) and Ame7 (bottom), making up the class M e l . €3, Clone Ame6, making up the class Meí’. Shading indicates transcribed regions of Me7 and Meí’. Arrows indicate locations of start and stop codons. B, BamHI; E, EcoRI; H, HinDIII; S, Sacl; Xb, Xbal; and Xh, Xhol. C, Diagrammatic representation of the F. trinervia NADP-ME cDNA (Borsch and Westhoff, 1990) showing the regions encoding the chloroplast transit peptide (T.P.) and mature NADP-ME and the 3’ UTR. Arrowheads indicate approximate locations of oligonucleotide primers used in Southern blotting and PCR analysis: 1, MEMAT; 2, ME internal 5 ’ and ME internal 3’; 3, NADP-universal 5‘ and NADP-universal 3’; 4, ME term 5’ and ME term 3‘; and 5, Me7 3’ UTR 5’ and M e l 3 ’ UTR 3’. Note that A and B are drawn to a different x a l e than C.

photosynthesis), no further characterization of Me3 was performed.

The two clones in class Mel (Amel and Ame7) and the single clone from Me2 (Ame6) all contained inserts of approximately 14 kb (Fig. 1, A and B). A 4-kb region of apparent overlap was identified by restriction mapping of hmel and Ame7. We confirmed the authenticity of this overlapping region by sequencing 335 bp at the 3’ end of Amel and the corresponding region of Ame7. The sequences of the two clones in this region were identical and corresponded to the central region of the F . trinervia cDNA (data not shown).

Based on the results of the above-mentioned Southern hybridization, the 5’ regions of M e l and Me2 were mapped to a 2.9-kb BamHI fragment of Amel and a 2.9-kb EcoRI fragment of Ame6, respectively. The 3’ ends of Mel and Me2 were mapped in Ame7 and Ame6 to the positions shown in Figure 1, A and B, using PCR with various restriction fragments of the genomic clones as templates and a pair of primers (referred to as ME term 5‘ and ME term 3 ’ ) based on the region of the F. trinervia cDNA encoding the carboxyl terminus of NADP-ME (Fig. 1C). Finally, Southern blotting using the partia1 cDNA Ame11 as a probe and PCR amplification with two pairs of primers corresponding to internal regions of the F . trinervia NADP-ME cDNA (Fig. 1C) were used to verify that all of the NADP-ME genes were present in Amel plus Ame7 and in Ame6 (data not shown).

Sequence Analysis of Mel and Me2

Because our ultimate goal was to study the factors reg- ulating the expression pattern of the C, isoform of NADP- ME, we needed to ascertain which gene encoded this protein. We considered that the 5’ end of the NADP-ME-

coding region would be most useful in identifying the correct gene, since this region would encode the chloroplast transit peptide and should therefore allow us to dis- criminate between genes encoding cytosolic and chloroplastic enzymes. Furthermore, for our planned promoter analysis (to be reported elsewhere), we were interested in mapping potential regulatory elements present in the upstream and downstream regions of the gene. For these reasons, we concentrated our further characterization of the F . bidentis genomic clones on the 5’ and 3‘ regions of Mel and Me2.

The 2.9-kb BamHI restriction fragment of Amel, which contained the 5‘ end of M e l , was subcloned and completely sequenced in both strands. Comparison of this sequence with those of the F. trinervia and F. pringlei cDNAs allowed the identification of exons 1 and 2 and introns 1 and 2, as shown in Figure 2A. The remaining DNA in this subclone (2120 bp) was located upstream of the NADP-ME start codon. The amino acid sequence inferred from exons 1 and 2, of Mel was very similar to the amino-terminal regions of F. trinervia and F. pringlei NADP-MEs, and contained an apparent chloroplast transit peptide of the same length as the F . trinervia and F . pringlei enzymes (Fig. 2C).

The corresponding region of Me2 was sequenced using the subcloned 14-kb insert of Ame6 as a template. Approx- imately 1.6 kb at one end of this subclone was sequenced in both directions. As with M e l , exons and introns were identified by comparison with the published cDNA sequences (Fig. 2B). Exons 1 and 2 of Me2 were found to be the same lengths as those of M e l , and the inferred protein was very similar to that encoded by Mel and the published cDNA sequences, including an apparent chloroplast transit peptide (Fig. 2C).

Detailed comparison of the inferred amino-terminal sequences of the two F. bidentis NADP-ME genes with those



Two Cenes for Chloroplastic NADP-Malic Enzyme in Haveria 1255

A Mel 5’ upstream DNA

2120bp exonl 186bp exon 2 30 bp 290 bp

Me2 5’ -- upstream DNA intron 1 intron 2

898bp exon 314 bp exon 2 30 bp 290 bp

C F. trinervia .............. T., .......... .., .... .=. ............ s.. .......................... ., ................. ”. ......... F.b,d.Mel H T S L L I S S ~ . L B R S S V I G G S R ~ Q ~ Q ~ ~ R ~ ~ V ~ ~ ~ V ~ ~ S M ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ V ~ V S V ~ ~ A V ~ ~ V ~ A ~ V A V ~ V ~ ~ S V G ~ ~ ~ ~ A V ~ ~ ~ ~ V ~ ~ ~ ~ G ~ ~ ~ ~ ~ ~ ~ Q ~ ~ ~ ~ ~ ~ V ~ V A

F.brd.Me2 M M S L N T S S V V R S S I S G V S R T Q S Q S V R ~ S V ~ ~ ~ M ~ ~ A ~ V N S N G ~ ~ ~ ~ S V ~ V ~ V ~ ~ ~ V R D V N ~ P V ~ V ~ V A ~ ~ ~ ~ ~ ~ ~ T A V V G G G V ~ ~ V ~ ~ ~ ~ ~ ~ ~ ~ ~ H F ~ T P W S V S V A I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

F,p,j”g,ej ..... 8 ............ W......................................................~P...............................

Figure 2. Comparison of the 5’ regions of Me7 and Me2. A and B, Diagrammatic representations of the sequenced 5 ‘ regions of Me7 and Me2, respectively. C, Comparison of the inferred amino acid sequences of the amino-terminal regions of NADP-ME from F. trinervia (top line), F. bidentis (F. bid.) Me7 (second Iine), F. bidentis Me2 (third line), and F. pringlei (bottom line). Vertical bars indicate identity between F. bidentis M e l and Me2. Dots indicate identity between F. trinervia NADP-ME and F. bidentis Me7 (top line) or F. pringlei NADP-ME and F. bidentis Me2 (bottom line). Vertical arrow indicates proposed transit peptide cleavage site in F. trinervia NADP-ME (Borsch and Westhoff, 1990). The sequences of the 5’ regions of Me7 and MeZwi l l appear in the GenBank database under accession nos. U44920 and U44922, respectively.

of the F. trinervia and F. pringlei NADP-ME cDNAs revealed some surprising similarities and differences. AI1 four of these sequences contained apparent chloroplast transit peptides of the same length. As shown in Figure 2C, the four sequences exhibited significant levels of amino acid identity. However, closer inspection revealed that the amino-terminal region of ME1 was much more similar to that of F. trinervia NADP-ME than to either ME2 or F. pringlei NADP-ME. Furthermore, the amino-terminal region of ME2 was much more similar to that of F . pringlei NADP-ME than to either ME1 or F. trinervia NADP-ME.

Analysis of the 3’ ends of Mel and Me2 confirmed the similarity of Mel to the F. trinervia cDNA and Me2 to the F. pringlei cDNA. To sequence the 3’ ends of both genes, an oligonucleotide primer (termed ME 3’ universal) was designed based on a conserved region of the F. trinervia and F. pringlei cDNAs just upstream of the stop codon. The region encoding the carboxyl terminus of NADP-ME and the region immediately downstream of the stop codon of Mel and Me2 were sequenced using this and several other primers. As shown in Figure 3A, the nucleotide sequences of the 3’ ends of the coding regions of the two genes were nearly identical. However, the regions downstream of the M e l and Me2 stop codons were quite different. Compari- son of the 3’ UTRs of the two F. bidentis genes with the F. trinervia and F. pringlei NADP-ME cDNAs demonstrated the striking similarity of Mel to the F. trinervia 3’ UTR (Fig. 3B) and Me2 to the F. pringlei 3’ UTR (Fig. 3C). Further- more, comparison of the 3’ UTR of the reported partia1 NADP-ME cDNA from the C,-C, intermediate species F . linearis (Rajeevan et al., 1991) with those of Mel and Me2 indicated that the F. linearis cDNA, like the F. pringlei cDNA, was much more similar to Me2 than to Mel (data not shown).

Genomic Southern Blot Analysis of NADP-ME Cenes Present in F. bidentis, F. trinervia, and F. pringlei

The high degree of similarity of the proteins encoded by the F. pringlei and F. trinervia NADP-ME cDNAs, along

with several other factors, led Lipka et al. (1994) to con- clude that these two cDNAs represent orthologous genes (i.e. descending from a common ancestral gene through speciation). However, our comparisons of the 5’ and 3’ regions of these cDNAs to F. bidentis Me l and Me2 seemed to indicate that the F. pringlei and F. trinervia cDNAs might actually represent paralogous genes (i.e. arising by gene duplication in a common ancestral species). If this were the case, it would seem likely that F. pringlei and F . trinervia would contain previously undetected NADP-ME genes representing Mel and Me2, respectively. To test this hypothesis, we used Southern blotting of genomic DNA isolated from these two species plus F. bidentis to determine the number and nature of NADP-ME genes present in each species.

To determine the total number of NADP-ME genes present in these three species, we designed a probe that was predicted to detect a11 NADP-ME genes while not detecting similar NAD-ME genes. Comparison of the inferred amino acid sequences of several plant NADP-MEs, including both cytosolic and chloroplastic isoforms, and a mitochondrial NAD-ME from Amaranthus (Long et al., 1994) revealed a 70-amino acid region that was highly conserved in both cytosolic and chloroplastic NADP-MEs but not in Amaranthus NAD-ME: DFANHNAFDLLEKY-T-

LGAGEAGTGIAEL (where dashes indicate positions for which no consensus could be determined). Comparison of the corresponding nucleotide sequences for this region showed that cytosolic and chloroplastic NADP-ME cDNAs differed by approximately 20%, whereas the Amaranthus NAD-ME cDNA was more than 50% different from the NADP-ME sequences. Based on these differences, we concluded that a DNA probe corresponding to this conserved region of NADP-ME would, under low-stringency hybridization and washing conditions, detect a11 plant NADP-ME genes and would be unlikely to detect NAD-ME genes. Furthermore, the relatively short length of such a probe would minimize the possibility that target sequences in the

HLVFNDDIQGTASVVLAGLL-ALKLVGGTLAD --HTFLF -



1256 Marshall et al. Plant Physiol. Vol. Ill, 1996

B

ATOTACAOCCCCAAATACCSCATCTATCSTTAAGTTTAGCGGGGGAAAAAI I I I I I II I I I II I I I I I I I I I I I I I I I I I I I IATOTACAOCCCCAAATACC9CAACTACC3TTAAACTT. ............

GGACAGTTGATCTGTTGCTGTTTGCAATT. TTTTAAAGGGTATGTTGTCAI I I I I N N I I I I I I I I I I I I I I I I I I I I I I I I I I

. TACGGTCAATCTGATACTGATAGCAATTATTITAGAGGATATTTTGGTA

GATGCATGTrGTAATGCTTGTTCATCAACACATTATATGACTTGCAGTTGI I I I I I I I I I I I I I I I I I I I I I I

TTTC. . .GTTG.GG'rccGTGTT. ..... .GTAATATTAGGGATGCTGTGT

CTGATGATGGAAACTrAAAGCTTAATACTACTTTrGTTTATTCACTTACAI I I I I I I I I I I I I I I I I I I

GTG. . . . TGTTrrCATAAACACAAGTATAAGTGAAATTGCAATTCTTTGC

AATACCGGTTGGGTTCTTTGTTTATCAGGAATGCTCATTGTATGTAGCTAI I I I I I I I I I I I I I I I I I I I

CAGACCTTTAMTTCCAATCAAGAAAAAGAATATGTAGCATGATCAGCTC

AAAGCTGGCCGTTTATAGTTTTATTGCCCTAAI I I I I I I I I I I IAACTTAG..... TTGTCAAGTCATTAGAAAAA

ATGTACAaCCCCAUTACCaCATCTATCaTTAAGTTTAGCOGGGGAAAAAI I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I IATQTACAacCCCAAATACCaCATCTACCOTTAAGTTTAGC. GGGAAAAAA

GaACAGTTGATCTGTTGCTOTTreCAATTnTTAAAGGGTATGTTSTCAGI I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

AGACAGTTGATCTGTTGCTGTGTX3CAATTTTTTAAAGGGTATGGTCTCAG

ATGCATGTTGTAATGCTTGTTCATCAACACATTATATGACTTGCAGTTGCI I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I IATGCATGTTGTAATGCTTOTTCATCAACACATTATATGACTTGCAGTTGC

TGATGATCGAAACTTAAAGCTTAAT. ACT. ACTTTTGTTTATT. CACTTAI I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I II I I II ITGATGATGGAAACTTAAAGCTTAATOACTGACTTrrGTTTATTGCACTGA

CAAATACCGGTTGGGTTCTTrGTTTATCAGGAATGCTCATrGTATGTAGCI I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I ICAAATACCGGTTGGGTTCTT. . GTTATCAGGAATGCTCATTGTGTGTAGC

TAAAAGCTGGCCG........ TrTATAGTTTTATTGCCCTAAAI I I I I I II M I I I I I I I I I I I I I I I I I I I I I I I I ITAAAAGCTGGCCGTTTATAGTTTTATAGTTTTArrGCCCTAAA

ATOTACAOCCCCAAATACCOCAACTACCOTTAAACT1TACGGTGAATCTGI I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I IATOTACAOCCCCAAATACCOCAACTACCOTTAAACTTTACGGTGAATCTG

ATACTGATAGCAATTATTTTAGAGGATATTTTGGTATTTGGTTGGGTGCG

ATACTGATAGCAATTATTTTAaAGGATATTTTGGTATTTGGTrAGGTGCG

TGTTGTAATATTAGGGATGCTGTGTGTGTGTrrTCATAAACACAAGTATAI I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I ITGTTGTAATATTAGGCATGCTGTGTGTGTGTTTTCATAAACACAAGTATA

AGTGAAATTGCAATTCTrrGCCAGACCTTTAAATTCCAATCAAGAAAAAGI I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I II I IIAGTGAAATTGCAATTCTTTGCC. GACCTATAAATTCCAATCAAGAAAAAA

AATATGTAGCATGATCAGCTCAACTTAGTTGTCAAGTCATTAGAAAAAI I I I I I I I I I I I I I I I I I I I I I II I I I I I I I M I I I I I I I I IAATATGTAGCATGA. .... . CAACTTAGTTGTCAAGTCATTAGAAAAA

F. bid. Mel

F. bid. Me2F. bid. Mel

F. bid. Mel

F. bid. HalF. bid. Me2r. bid. MelF. bid. Me2

F. bid. Mel

F. bid. Me2

F. bid. Mel

F. bid. Me2

F. bid. Mel

F. trinervia

F. bid. MelF. trinervia

F. bid. Mel

F. trinervia

F. bid. Mel

F. trinervia

F. bid. Mel

F. trinervia

F. bid. Mel

F. trinervia

F. bid. Me2

F. pringlei

F. bid. M»2

F. pringleiF. bid. MelF. pringlei

F. bid. Me2

F. pringleiF. bid. Me2F. pringlei

Figure 3. Comparisons of the nucleotide sequences of the 3' regionsof F. bidentis (F. bid.) Mel and Me2 with those from the F. trinerviaand F. pringlei NADP-ME cDNAs. Comparisons were made using theGenetics Computer Group (Madison, Wl) program, Gap, with gapcreation penalty = 2 and gap extension penalty = 0.2. Codingregions and stop codons are shown in boldface. Vertical bars indi-cate identical nucleotides. A, F. bidentis Mel versus F. bidentis Me2.B, F. bidentis Me1 versus F. trinervia. C, F. bidentis Me2 versus F.pringlei. The sequences of the 3' regions of Mel and Me2w\\\ appearin the GenBank sequence database under accession nos. U44921and U44923, respectively.

genomic DNA would contain sites for the restriction en-zymes used in the Southern analysis. Therefore, the num-ber of bands hybridizing to this probe would be morelikely to reflect the actual number of NADP-ME genespresent.

We designed a pair of oligonucleotide primers (termedNADP-universal 5' and NADP-universal 3', Fig. 1C) thatflanked the conserved region of NADP-ME and amplifiedthis region by PCR using the F. bidentis NADP-ME cDNAAmell as template. The resulting 214-bp PCR product

(termed NADP-universal) was radiolabeled and used toprobe Southern blots of F. bidentis, F. pringlei, and F. tri-nervia genomic DNA under low-stringency conditions. Asshown in Figure 4, NADP-universal detected two stronglyhybridizing bands and two weaker bands in all lanes ofblots of F. bidentis and F. trinervia DNA. The relative mo-bilities of the strongly hybridizing bands detected in F.bidentis DNA matched those predicted from the restrictionmaps of the clones representing Mel and Me2 (Fig. 1, A andB). The two weaker bands present in each lane of F. bidentisDNA could be selectively removed by washing at higherstringency (data not shown), indicating that they were lesssimilar to the probe and probably represented distantlyrelated NADP-ME genes. Based on these results, we con-cluded that both F. bidentis and F. trinervia contained atleast three, possibly four, NADP-ME genes.

It is interesting that approximately eight bands wereseen in all lanes when F. pringlei DNA was probed withNADP-universal (Fig. 4). We suspected that the larger ap-parent number of NADP-ME genes present in F. pringleicould be related to ploidy, since some collections of thisspecies have been reported to be tetraploid (n = 36),whereas all other Flaveria spp. so far analyzed are diploid(n = 18; Powell, 1978). To test this hypothesis, we deter-mined the chromosome number of the strains of F. pringleiand F. bidentis used in our laboratory. Root tip cells of F.pringlei were found to contain 72 chromosomes, and thoseof F. bidentis contained 36 chromosomes (data not shown).

To determine the number of copies of Mel present in thethree species, Southern blots were probed with an Mel-specific DNA fragment under high-stringency conditions.This 234-bp probe, which was made by PCR using a sub-

E E+S H E E+S H E E+S H

F. bidentis F. pringlei

Figure 4. Southern blots of genomic DNA from three Flaveria spp.cut with three restriction enzymes and probed with NADP-universal.E, fcoRI; E + S, fcoRI plus Sacl; and H, H/ndlll. Solid and openarrowheads indicate bands predicted from the restriction maps of theclones in classes Mel and Me2, respectively. Numbers at left indi-cate sizes in kb. The three panels were run on one gel, transferred toa single membrane, and probed simultaneously.



Two Genes for Chloroplastic NADP-Malic Enzyme in Flaveria 1257

cloned restriction fragment of Ame7 as a template and theoligonucleotide primers Mel 3' UTR 5' and Mel 3' UTR 3',extended from the NADP-ME stop codon to just upstreamof the region corresponding to the beginning of the poly(A)tail of the F. trinervia NADP-ME cDNA (Borsch andWesthoff, 1990). As shown in Figure 5, single bands weredetected in F. bidentis and F. trinervia DNA digested withEcoRI or EcoRI plus Sad. The detection of two Mel bands inHwdlll-digested DNA from these species can be explainedby the presence of a Hmdlll site just downstream of thestop codon of both F. bidentis Mel and the F. trinerviaNADP-ME cDNA. The relative mobilities of the bands seenin F. bidentis DNA matched those predicted by restrictionmapping of Ame7 (Fig. 1A). Three Mel bands were detectedin all lanes containing F. pringlei DNA (Fig. 5).

The number of Me2 genes present in each species wasdetermined by probing Southern blots with an Me2-specificDNA fragment under high-stringency conditions. This209-bp probe represented the 3' UTR of Me2 and was madeby PCR using a pair of primers derived from the F. pringleicDNA (termed Me2 3' UTR 5' and Me2 3' UTR 3') and theinsert of Ame6 as a template. As shown in Figure 6, singleMe2 bands were detected in all lanes containing F. bidentisor F. trinervia DNA. The relative mobilities of these bandswere distinct from those detected with the Mel -specificprobe and, in the case of F. bidentis, corresponded to thesizes predicted from restriction mapping of Ame6 (Fig. IB).As was the case with the Mel -specific probe, three stronglyhybridizing bands were detected in lanes containing F.pringlei DNA. One band in each of the F. pringlei lanesappeared to correspond to a band detected by the Mel-specific probe (Fig. 5), indicating that some cross-hybrid-

E E+S H E E+S H E E+S HKb

E E+S H E E+S H E E+S H

6.4-6.6-

2.3 -2.0-

F. bidentis F. pringlei F. trinervia

Figure 5. Southern blots of genomic DNA from three Flaveria spp.probed with a radiolabeled PCR product representing the 3' UTR ofMet. Lane designations and size markers are the same as those inFigure 4. Arrowheads indicate bands detected in F. pringlei DNA thathad the same relative mobility as bands detected using a probespecific for Me2 (see text and Fig. 6). The blot used for this figure wasthe same as that used for Figures 4 and 6.

9.4- <«•>

6.6-

23-2.0-

0.66-

F. bidentis F, pringlei F. trinervia

Figure 6. Southern blots of genomic DNA from three Flaveria spp.probed with a radiolabeled PCR product representing the 3' UTR ofMe2. Lane designations and size markers are the same as those inFigures 4 and 5. Arrowheads indicate bands detected in F. pringleiDNA that had the same relative mobility as bands detected using aprobe specific for Me1 (see text and Fig. 5). The blot used for thisfigure was the same as that used for Figures 4 and 5.

ization might have occurred with one of the NADP-MEgenes in this species. However, at least two bands uniqueto either the Mel- or Me2-specific probe were seen in eachF. pringlei lane.

Only Me1 Is Highly Expressed in Flaveria Leaves

Because Mel and Me2 encode very similar chloroplasticforms of NADP-ME, it seemed possible that either or bothof their products could be utilized in C4 photosynthesis. Asa first step in determining which of the two genes washighly expressed in photosynthetic tissue, we purifiedNADP-ME from F. bidentis leaves and determined itsamino-terminal amino acid sequence. The sequence NH2-GGVEDMYGEDTATEDQYITP precisely matched that pre-dicted from Mel and differed from the inferred amino acidsequence of Me2 in four positions (Fig. 2C). In none of thesepositions was a secondary peak corresponding to theamino acid predicted for Me2 detected, indicating that F.bidentis leaf NADP-ME does not contain a significant pro-portion of the Me2 product. The amino terminus of ME1did not correspond to the transit peptide cleavage sitepredicted by Borsch and Westhoff (1990), but rather was 18residues downstream (Fig. 2C). This would seem to indi-cate that either transit peptide cleavage occurs at a site notpredicted by the rules of Gavel and von Heijne (1990) or theprotein is proteolytically modified after transit peptidecleavage or during purification (see "Discussion").

To further clarify which NADP-ME gene is utilized in C4photosynthesis, we examined Mel and Me2 mRNA levelspresent in leaves of seven Flaveria spp. Previous analyses ofNADP-ME mRNA in Flaveria leaves and other organs re-lied on probes that included part or all of the coding region



1258 Marshall et al. Plant Physiol. Vol. 111, 1996

of the F. trinervia or F. pringlei cDNAs (Borsch andWesthoff, 1990; Rajeevan et al., 1991; Lipka et al., 1994). Inthese latter studies, the high degree of similarity of theNADP-ME-coding regions would have allowed the deter-mination of the total amount of NADP-ME mRNA present;however, the probes used would not have discriminatedbetween mRNA derived from Mel- or Me2-type NADP-MEgenes. We have used Mel- and Me2- specific probes toaddress the question of which NADP-ME genes are uti-lized in C4 photosynthesis in a range of Flaveria spp. Thisquestion was of particular interest because C4 photosyn-thesis is believed to have evolved at least twice in Flaveria(Powell, 1978), and it seemed possible that differentNADP-ME genes could have been utilized in C4 photosyn-thesis in these two events.

Total RNA was isolated from leaves of F. bidentis and sixother Flaveria spp. These included a C4 species (F. trinervia),two C4-like C3-C4 intermediate species (F. palmeri and F.brownii), two other C3-C4 intermediates (F. floridana and F.linearis), and a C3 species (F. pringlei). The level of totalNADP-ME mRNA present in leaves of these species wasdetermined using northern blots with the non-gene-specific probe NADP-universal. As expected from previousstudies, NADP-ME mRNA levels approximately paralleledthe degree of C4 photosynthesis exhibited by the variousFlaveria spp., with no NADP-ME mRNA being detectablein total RNA isolated from leaves of the C3 species F.pringlei (Fig. 7A).

When an Mel -specific probe derived from the 3' UTR ofthe F. bidentis Mel gene was used, Mel mRNA was detected

<(' <(' <(' <C" <(' <(' <('

B

Figure 7. Northern blot analysis of NADP-ME gene expression inleaves of seven Flaveria spp. A, Total NADP-ME mRNA (probed withNADP-universal). B, Met mRNA (probed with /Vie/-specific 3' PCRproduct made using the F. bidentis partial cDNA, Ame11, as tem-plate). C, Mel mRNA (probed with /Vle7-specific 3' PCR productmade using F. brownii genomic DMA as template). D, Me2 mRNA(probed with Me2-specific 3' PCR product made using F. browniigenomic DNA as a template. This result is essentially identical withthat obtained when an Me2-specific probe from F. bidentis wasused). All lanes contained approximately 10 /j.g of total RNA.

Figure 8. Size dimorphism in 3' UTRs of NADP-ME present in sevenFlaveria spp. Genomic DNA from seven species was used as tem-plate in PCR amplifications using a pair of primers based on the 3'UTR of F. bidentis Mel. Far right lane (MW) contains DNA sizemarkers in integral multiples of 100 bp.

only in total RNA isolated from leaves of F. bidentis, F.trinervia, and F. palmeri (Fig. 7B). This probe did not detectany Mel mRNA in total RNA isolated from F. brownii, F.floridana, or F. linearis, even though these species showedsignificant amounts of total NADP-ME mRNA when ana-lyzed using NADP-universal (Fig. 7A). To test whether thefailure to detect Mel mRNA in these species might be dueto sequence differences in the 3' UTRs of the Mel genes, wemade a second Mel -specific probe by PCR amplification ofF. brownii genomic DNA using two oligonucleotide prim-ers flanking the Mel 3' UTR. When northern blots wereanalyzed with this probe, Mel mRNA was detected in alllanes except that containing F. pringlei RNA (Fig. 7C).

When 10 fj,g of total RNA from the seven Flaveria spp.were analyzed using an Me2-specific probe from either F.bidentis or F. brownii, no Me2 mRNA was detected in any ofthe samples (Fig. 7D). In both cases, a small amount of theunlabeled Me2 3' PCR product was included on the blot asa positive control to verify that hybridization and washingconditions were appropriate (data not shown).

The 3' UTRs of the Mel genes present in the sevenFlaveria spp. were further characterized using PCR withgenomic DNA as templates. As shown in Figure 8, a sizedimorphism in the Mel 3' UTR was found between F.bidentis, F. trinervia, F. palmeri, and F. pringlei on the onehand, and F. brownii, F. floridana, and F. linearis on theother. Preliminary sequence analysis of the 3' UTRs of Melfrom F. brownii, F. linearis, and F. floridana indicated that thebasis for this dimorphism was a 25-bp deletion 32 bpdownstream of the stop codon (J.S. Marshall, unpublisheddata). The Mel 3' UTRs of these three species are otherwisevery similar to those of F. bidentis and F. trinervia. Theoccurrence of this dimorphism parallels the phylogenetic




Me1

Me2

actin

Figure 9. Northern blot analysis of Me! and Me2 expression usingpoly(A)+ RNA. Poly(A)+ RNA isolated from 500 jj.g of F. bidentis leafor root total RNA was probed using Mel-, Me2-, or actin-specificPCR products.

grouping of 20 Flaveria spp. given by Powell (1978) basedon morphological, experimental, chemical, and eco-geographic considerations. In this scheme, the genus Fla-veria is divided into two major lineages: one including(among others) the species F. bidentis, F. trinervia, F. palmeri,and F. pringlei, and the other including (among others) F.brownii, F. floridana, and F. linearis.

Me2 Is Expressed at a Low Level in Leaves andRoots of F. bidentis, whereas Me1 Transcript IsPresent Only in LeavesTo determine whether Mel expression is restricted to pho-tosynthetic tissue and whether Me2 is expressed at all,poly(A)+ RNA was isolated from 500 ju,g of F. bidentis leafor root total RNA and probed on northern blots using Mel-and Me2-specific probes. As shown in Figure 9, Mel washighly expressed in leaves but not in roots. No Mel mRNAwas detectable in poly(A)"1" RNA isolated from roots, evenon prolonged exposure of the blot to the Phosphorlmagerscreen (Molecular Dynamics, Sunnyvale, CA). In contrast,Me2 mRNA was detectable at low, but approximately sim-ilar, levels in poly(A)+ RNA from both leaves and roots(Fig. 9). The signals for Me2 mRNA shown in Figure 9 werequantitated and normalized using actin mRNA as a stan-dard.2 After normalization to actin levels, Me2 mRNA wasfound to be twice as abundant in leaves as in roots. Incontrast, after normalization, the Mel mRNA level was atleast 200-fold greater in leaves than in roots.

Light Regulation of Me1 Expression in F. bidentis LeavesBecause leaf-specific Mel is involved in C4 photosynthe-

sis, it seemed reasonable to expect that its level of expres-sion might be regulated by light. We therefore isolated leaf

2 As noted, poly(A)+ RNA loads were based on yields fromequivalent amounts (500 p,g) of leaf and root total RNA. It ispossible that the difference seen in the amount of actin mRNAbetween these samples (3.4-fold) is due, in part, to an overestima-tion of the amount of cytosolic RNA present in leaf total RNAcaused by the large amount of chloroplastic rRNA present.

total RNA from greenhouse-grown F. bidentis plants, fromgreen plants that had been placed in darkness for 2 or 5 d,and from green plants placed in darkness for 5 d and thenreturned to light for 2, 4, 8, or 24 h. The RNA was analyzedon northern blots using a 3' Mel-specific probe. As shownin Figure 10, Mel mRNA declined to an almost undetect-able level after 5 d in darkness. Levels of Mel mRNAincreased noticeably after 2 h in light and returned to thelevel present in greenhouse-grown leaves between 8 and24 h after transfer back to light.

DISCUSSION

We have identified and characterized genomic clonesrepresenting two distinct NADP-ME genes, Mel and Me2,from the C4 plant F. bidentis. Sequence analysis of theseclones indicates that the two genes encode very similarproteins that contain predicted chloroplast transit peptidesof the same length. Southern blot analysis of F. bidentisgenomic DNA using NADP-universal, a probe expected tohybridize to any plant NADP-ME gene, demonstrated thatthis species contains a minimum of three, possibly four,NADP-ME genes. Comparison of the relative mobilities ofbands detected on Southern blots with restriction frag-ments predicted from mapping the clones representingMel and Me2 allowed us to positively assign two of thefour bands present in each lane to either Mel or Me2. Giventhat cytosolic NADP-MEs have been reported from a di-versity of plant species and that only chloroplastic andcytosolic NADP-MEs have been identified in plants, itseems likely that the remaining two NADP-ME genes in F.bidentis encode cytosolic isoforms of the enzyme.

We were surprised to find two very similar genes forchloroplastic NADP-ME in F. bidentis, since previous work-ers had suggested, based largely on genomic Southernblotting, that the cDNA clones isolated for chloroplasticNADP-ME of F. trinervia (C4) and F. pringlei (C3) representunique orthologous genes in these species (Lipka et al.,1994). However, comparison of the 5' and 3' regions of thetwo F. bidentis genes with the NADP-ME cDNAs isolatedfrom F. trinervia and F. pringlei demonstrated that thesecDNAs were more similar to Mel and Me2, respectively,than to each other. This suggests that the F. pringlei and F.trinervia cDNAs actually represent paralogous genes and

•c-4?

C? C?

*^^^7

Figure 10. Me1 expression is light-regulated in F. bidentis. TotalRNA was isolated from leaves of F. bidentis plants grown in thegreenhouse (light grown) and after transfer to darkness for 2 or 5 d,followed by return to continuous light for 2, 4, 8, or 24 h. Approx-imately 10 jLig of total RNA were loaded per lane.



1260 Marshall et al. Plant Physiol. Vol. 11 1 , 1996

that other genes for chloroplastic NADP-ME might be present in these two species. Southern blotting of genomic DNA from F. bidentis, F . pringlei, and F . trinervia using probes specific for Mel and Me2 confirmed that these species a11 contained representatives of both Mel and Me2.

We believe that the disparity between our Southern blot analyses and those of Lipka et al. (1994) is due to differences in the probes used and the stringency of hybridization and washing. The probes we used were relatively short, and the presence of restriction sites in the corresponding F. bidentis genomic target DNA was known from restriction digestion of the clones isolated for Mel and Me2. In addition, we used relatively low-stringency conditions of hybridization and washing to allow detection of a11 NADP-ME genes. In contrast, the probes used by Lipka et al. (1994) were relatively long and their genomic DNA target sequences were uncharacterized. Furthermore, their hybridization and washing conditions were relatively stringent, apparently precluding the detection of anything but nearly identical sequences. We have made NADP- universal probes by PCR using both Mel and Me2 as templates and compared their hybridization patterns on South- ern blots of F . bidentis DNA under stringency conditions similar to those used by Lipka et al. (1994). Under these conditions, bands corresponding to the gene from which the probe derived were quite intense, whereas those corresponding to the reciproca1 gene were relatively faint (J.S. Marshall, unpublished data). Based on these results, we believe that the hybridization and washing conditions used by Lipka et al. (1994) were too stringent to allow detection of bands corresponding to Mel in F . pringlei DNA and Me2 in F. trinervia DNA, since only Me2-derived probes were used with F . pringlei DNA and only Mel-derived probes were used with F. trinervia DNA. It would be interesting to compare the patterns of bands shown in figure 3 of Lipka et al. (1994) with those obtained using their F. pringlei (Me2) probes with F. trinervia DNA and their F. trinervia ( M e l ) probes with F . pringlei DNA.

The similarity of Mel and Me2 is striking. As noted earlier, the products of both genes include putative chloroplast transit peptides of the same length and very similar composition (Fig. 2C). Furthermore, based on comparison of the cDNAs from F. trinervia and F . pringlei, the inferred amino acid sequences of the mature NADP-ME products of Mel and Me2 are even more similar (>90%) than those of the chloroplast transit peptides (Lipka et al., 1994). Finally, the placement of introns 1 and 2 is identical in Mel and Me2 from F . bidentis. These facts would seem to indicate that the two genes are closely related to one another and arose relatively recently through gene duplication. The presence of both Mel and Me2 in a range of Flaveria spp., including members of both major branches of the genus, indicates that this duplication event probably either preceded or coincided with the evolution of Flaveria.

The expression patterns of Mel and Me2 may also provide some insight into the evolution of NADP-ME genes and C, photosynthesis in Flaveria. We have determined that Me2 is expressed at low but similar levels in photosynthetic and nonphotosynthetic tissues of F. bidentis (Fig. 9). In

contrast, Mel is highly expressed in leaves of the C, species F. bidentis and F. trinervia and is expressed in C,-C, intermediate species at levels corresponding to the degree of C, photosynthesis exhibited by those species (Fig. 7, B and C). No Mel expression was observed in leaves of the C, species F . pringlei, a fact that is corroborated by the isolation of 10 independent Me2 cDNAs and no Mel cDNAs from a F. pringlei leaf cDNA library, even though the probe used was from a Mel cDNA (Lipka et al., 1994). Mel expression is light-regulated (Fig. 10) and leaf-specific; no Mel mRNA could be detected in poly(A)+ RNA isolated from F. bidentis roots (Fig. 9). Taken together, these facts would seem to indicate that the product of Me2 is a "housekeeping enzyme" that is required for some nonphotosynthetic process in both C, and C, plants. This enzyme probably corre- sponds to the plastid-localized NADP-ME activity mea- sured in the C, plants marrow and soybean by El-Shora and ap Rees (1991). M e l , on the other hand, appears to be strictly involved with C, photosynthesis, since its expression leve1 correlates with C, photosynthesis in Flaveria, and no expression is seen in roots.

We believe that ME2 probably represents an ancestral form of plastid-localized NADP-ME that may be present in a11 plants, and that ME1 is the product of a recent gene duplication that has been specifically adapted for C, photosynthesis in Flaveria. It would be interesting and infor- mative to study the NADP-ME genes of closely related genera such as Sartwellia and Haploestkes. These genera, which are the closest to Flaveria and exhibit more ancestral traits than Flaveria, are characterized by C, species (Powell, 1978). We might expect that both genera would contain representatives of Me2 but not M e l . It is possible that duplication of key C, enzymes such as NADP-ME and PEPCase is a distinguishing characteristic of Flaveria spp. Such duplications may have facilitated the evolution of C, photosynthesis in this genus by providing genes whose expression could be modified without adversely affecting other, perhaps critical, functions. Such a "pre-adaptation" favoring the evolution of C, photosynthesis might be inferred from the fact that C, photosynthesis appears to have arisen at least twice in Flaveria (Powell, 1978). It is interesting that no other genus in the Asteraceae has been found to contain both C, and C, species (Smith and Turner, 1975).

We have discovered that the NADP-ME gene family of Flaveria is more complex than previously recognized. In addition, we have, through the use of protein purification/ sequencing and mRNA studies, determined unequivocally which member of this family is involved in C, photosynthesis. This work has opened the door not only for the evolutionary studies mentioned above, but for studies of the factors determining bundle-sheath cell-specific expression of NADP-ME in C, leaves as well. We are currently in the process of dissecting the upstream and downstream regulatory regions of the Mel gene and studying their effects on the expression of reporter genes through the use of stably transformed F . bidentis plants (Chitty et al., 1994). Through this work, we are beginning to unravel the molecular events that led to the development of C, photosynthesis in Flaveria.




ACKNOWLEDCMENTS

We thank Maurice Ku and Susanne von Caemmerer for providing plant material, Tim Nelson for a maize NADP-ME cDNA clone, John Mason for isolation of the Ame11 cDNA clone, and Tara Goodsell for cheerfully preparing the figures. Bob Furbank, Brian Surin, and Paul Whitfeld provided helpful comments con- cerning the manuscript. We are particularly grateful to Dick Brock for root tip chromosome counts and Tony Ashton for purification Of NADP-ME.

Received January 26, 1996; accepted April 29, 1996. Copyright Clearance Center: 0032-0889/96/111/ 1251 / 11.

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Genes Encode Highly Similar Chloroplastic NADP-Malic ...that Mel expression in leaves parallels the degree of C, photosyn- thesis in seven Haveria species. Furthermore, whereas Me2