INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Malic enzymes (MEs) play an important role in a number of metabolic processes that require the conversion of malate to NAD(P)H, pyruvate, and CO₂. In plants, these enzymes contribute to C₄ photosynthesis, pH balancing mechanisms, and fruit ripening processes. MEs also provide reducing agents and carbon skeleton to biosynthetic pathways, with the NAD-ME isoforms functioning predominantly in the mitochondria and the NADP-ME isoforms functioning in the cytoplasm and plastid (Wedding, 1989; Edwards and Andreo, 1992; Drincovich et al., 2001). With the exception of the isoforms acting in C₄ photosynthesis, the correspondence between the observed cellular ME isoforms and their metabolic roles remain unclear. The genus Flaveria presents an opportunity to determine this correspondence.

The genus Flaveria (Asteraceae) contains C₃ and C₄ species and C₃-C₄ intermediate species with C₄ photosynthetic capacity ranging from C₃- to C₄-like. These species can be grouped into two lines based on genetic hybridization experiments, the number of phyllaries (floral bracts), and molecular phylogenetic studies (Powell, 1978; Bayraktaroglu, 1993; Kopriva et al., 1996; Marshall et al., 1996). All C₃ and C₄ species, in addition to some C₃-C₄ intermediates, belong to the 3-4 phyllary line, and the remaining C₃-C₄ species make up the 5-6 phyllary line. These well-characterized relationships make it possible to compare ME isoform abundance and gene expression patterns among closely related species with distinct metabolic schemes that utilize ME activity.

In the genus Flaveria, NADP-ME is encoded by a small multigene family in each species. Only ChlMe1, which encodes the chloroplastic NADP-malic enzyme (CHLME) that acts in the C₄ cycle, is expressed at high level in leaves of C₄ species. ChlMe1 was cloned as a cDNA from Flaveria trinervia (C₄; Börsch and Westhoff, 1990; Rajeevan et al., 1991) and as genomic clones from Flaveria bidentis (C₄; Marshall et al., 1996) and F. trinervia (L. Lai and T. Nelson, unpublished data).A second gene (ChlMe2) encoding a plastidic NADP-ME has a non-leaf-specific expression pattern (Lipka et al., 1994; Marshall et al., 1996). The ChlMe1 and ChlMe2 genes are present and expressed in all Flaveria spp. regardless of photosynthetic type (Marshall et al., 1996; L. Lai, L. Wang, and T. Nelson, unpublished data).

Cytosolic NADP-ME (CYTME) has been described in several C₃ plants (Walter et al., 1988, 1990; van Doorsselaere et al., 1991; Franke and Adams, 1995; Knee et al., 1996) but not in C₄ plants. Here, we report the cloning of the CytMe cDNAs from Flaveria pringlei (C₃) and show that similar genes are present in C₄ and C₃-C₄ species of the same genus. Northern-blot analysis demonstrates that CytMe expression patterns are more complex in the C₄ species F. trinervia than in the C₃ species F. pringlei. This finding suggests that CytMe gene expression is regulated to accommodate C₄ photosynthesis, possibly to assure apportionment of the malate pool.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Isolation and Characterization of F. pringlei CytMe cDNAs

We screened a F. pringlei leaf cDNA library with a probe designed to detect both ChlMe and CytMe cDNAs, as described in "Materials and Methods." Of 5 × 10⁵ primary phagemids screened, six clones were isolated which correspond to two highly similar CytMe mRNAs. No ChlMe cDNAs were isolated, consistent with the low level of expression of ChlMe in F. pringlei fully expanded leaves (see below; L. Lai, L. Wang, and T. Nelson, unpublished data).

Complete sequences were obtained for CytMeA (2,044 bp; accession no. AF288920), represented by four clones and CytMeB (2,021 bp; accession no. AF288921), represented by two clones. CytMeA cDNA comprises an open reading frame of 1,769 bp, a 5'-untranslated region (UTR) of 82 bp, and a 3'-UTR of 156 bp; CytMeB cDNA comprises an open reading frame of 1,769 bp, a 5'-UTR of 41 bp, and a 3'-UTR of 191 bp. These two cDNAs are 97% identical at the nucleotide level and 98% identical at the amino acid level (data not shown). CytMeB contains two regions absent in CytMeA: (a) eight bp in the 5'-UTR and (b) 12 bp in the 3'-UTR (Fig. 1, A and B, respectively). These regions are part of short tandem repeats (half-arrows in Fig. 1), which might play a role in the regulation of mRNA processing, stability, or translatability. A common poly(A) site was found in most clones; however, one clone in each class was found to end at a position 18 bp further downstream. At least one poly(A) signal, highly similar to the consensus AATAAA motif, was found within 40 bp upstream of these poly(A) sites (Fig. 1B, bold, italicized regions). The near-duplicate genes represented by these cDNAs may be a consequence of the allotetraploid nature of F. pringlei.

View larger version (61K): [in this window] [in a new window]   Figure 1.   Alignment of the Flaveria spp. ChlMe and CytMe cDNA sequences. A, Sequences in the 5' regions. The solid underline indicates the ChlMe1 fragment used as the ChlMe-specific probe; the dashed underline indicates the CytMeA fragment used as the CytMe-specific probe. The 5' end of the short F. trinervia mRNA is indicated by an asterisk. B, Sequences in the 3' regions. The bold, italicized regions are the putative polyadenylation signals and the outlined nucleotide T indicates the upstream poly(A) addition site in the CytMe cDNAs. Identical nucleotides are highlighted with a gray background. Half-arrows indicate direct repeat sequences in the CytMe cDNAs. Boxes show the start codons in A and the stop codons in B. Ftr, F. trinervia; Fpr, F. pringlei.

CytMe Genes Are Highly Similar to ChlMe Genes

The CytMe cDNAs are 74% and 75% identical with the F. trinervia ChlMe1 cDNA (Börsch and Westhoff, 1990) and the F. pringlei ChlMe2 cDNA (Lipka et al., 1994), respectively, in the regions encoding the mature proteins (data not shown). They differ from ChlMe genes by the absence of regions encoding the plastidic transit peptide and by many differences in the 5'- and 3'-UTRs (Fig. 1). In the 3'-UTRs, they are only 29% to 36% conserved. Low conservation in the 3'-UTRs (39%) is also found between the two ChlMe cDNAs. At the deduced amino acid level, CYTME is 76% and 78% identical with the F. trinervia CHLME1 and F. pringlei CHLME2, respectively, in the mature protein regions (data not shown).

In comparison with other plant NADP-MEs (data not shown), sequence conservation was found in three regions that form the dinucleotide-binding folds of NAD- and NADP-linked oxidoreductases and in the five cysteines conserved in all plant NADP-MEs (e.g. see Lipka et al., 1994).

CYTME Is Encoded by a Small Multigene Family in C₃ Species but by a Single Gene in C₃-C₄ and C₄ Species

We found CytMe genes in all 10 Flaveria spp. examined. The species included were two C₃ species (F. pringlei and Flaveria robusta), six intermediate species (Flaveria linearis, Flaveria floridana, Flaveria brownii, Flaveria angustifolia, Flaveria anomala, and Flaveria vaginata), and two C₄ species (F. trinervia and F. bidentis). All species belong to the 3-4 phyllary line except for F. linearis, F. floridana, and F. brownii, which belong to the 5-6 phyllary line (Powell, 1978). For most of the species, the CytMe-specific probe (Fig. 1A) detected a single, strongly hybridizing fragment, suggesting they contain a single-copy gene (Fig. 2A). Multiple bands were detected in F. anomala, F. pringlei, and F. robusta, suggesting the presence of a small multigene (two-three copies) family in these species. To test whether the CytMe probe is specific for CytMe genes in all species, the DNA blots were reprobed with a ChlMe probe (Fig. 1A) that detects both ChlMe1 and ChlMe2 genes (Fig. 2B). The specificity of the probes is supported by the observation that they detect sets of bands with little or no overlap. The few apparently overlapping bands might be coincidental or attributable to the low-stringency hybridization conditions employed. However, the pattern obtained with each probe is unique.

View larger version (61K): [in this window] [in a new window]   Figure 2.   Southern analysis of genomic DNAs from Flaveria spp. using a CytMe-specific probe (A) and a ChlMe-specific probe (B). Both ChlMe1 and ChlMe2 are detected by the ChlMe-specific probe, resulting in multiple hybridizing bands in B. The species are grouped according to types of photosynthesis. Parentheses in each C3-C4 group indicate phyllary line. Within each of the C3-C4 groups, the species are placed in order of increasing C4 capabilities (Ku et al., 1991). Note that F. brownii and F. vaginata have been characterized as C4-like. RI, EcoRI; H3, HindIII; Xb, XbaI. Numbers at left indicate sizes in kilobase pairs.

CytMe Is Expressed in All Mature Organs of F. pringlei (C₃) and F. trinervia (C₄)

To determine whether the expression pattern of CytMe differs in leaves of C₃ and C₄ species, we compared CytMe mRNA levels in F. pringlei (C₃) and F. trinervia (C₄) and also examined the levels of both ChlMe1 and ChlMe2 mRNAs in toto (referred hereafter collectively as ChlMe mRNAs). Total RNA was isolated from leaves (fully expanded), stems, and roots of F. pringlei and F. trinervia and hybridized with the CytMe- and ChlMe-specific probes (Fig. 3). In F. pringlei, the patterns of CytMe and ChlMe mRNA accumulation are similar. Both mRNAs are found at comparable levels in stems and roots but accumulate to severalfold lower levels in leaves. In F. trinervia, CytMe mRNA was relatively uniform in abundance in all three organs, whereas ChlMe mRNA levels were extremely high in leaves compared with stems and roots. Based on the overall uniform expression of CytMe in all vegetative organs of both species, it is likely that CytMe plays a non-photosynthetic role in plant metabolism.

View larger version (100K): [in this window] [in a new window]   Figure 3.   Organ-specific accumulation of CytMe and ChlMe mRNAs in F. pringlei and F. trinervia grown in a growth room. To normalize RNA loading, the blot was stripped and rehybridized with a ubiquitin probe (Ubi). A photograph of the 25S rRNA bands from the ethidium bromide-stained gel is also shown. L, Fully expanded leaves; S, stems; R, roots. Approximately 20 µg of total RNA was loaded per lane.

Alternative CytMe Transcripts in F. trinervia Leaves Vary in Proportion during Leaf Development

Because CYTME and CHLME roles may change with the onset of photosynthetic competence, we compared CytMe and ChlMe gene expression patterns throughout leaf development in both the C₃ and C₄ Flaveria spp. In F. pringlei leaves, the level of CytMe mRNA increased slightly as the leaves matured (Fig. 4A). The deviations observed at 27% and 60% full-length stages might result from inadvertent stress and/or wounding in these leaf samples, because CytMe is responsive to these factors (Schaaf et al., 1995). ChlMe mRNA accumulation changed dramatically with leaf development, peaking early and then declining to a low and steady level.

View larger version (57K): [in this window] [in a new window]   Figure 4.   Changes in the levels of CytMe and ChlMe mRNAs during leaf development in F. pringlei (A) and F. trinervia (B). Leaves of various lengths representing different stages of maturity were collected from plants grown in a growth room. To normalize RNA loading, the blots were stripped and rehybridized with a ubiquitin probe (Ubi). Photographs of the 25S rRNA bands from the ethidium bromide-stained gels are also shown. Approximately 20 µg of total RNA was loaded per lane.

During leaf development in F. trinervia, distinct CytMe transcripts of apparent sizes 2.0 and 2.4 kb were present, depending on developmental state (Fig. 4B). In young leaves, the 2.4-kb transcript was predominant. From the 63% full-length stage onward, levels of the 2.0-kb transcripts increased. To determine whether these size variants were due to alternative splicing or to alternative 5' or 3' ends, we analyzed the CytMe mRNAs by reverse transcription (RT)-PCR and RACE, as described in "Materials and Methods." RT-PCR with a variety of primers revealed no size variation within the coding portions of the 2.0- and 2.4-kb mRNAs, suggesting they do not differ by alternative splices. By means of 3' RACE assays, we found small variations in the poly(A) site that were insufficient to account for the size difference (Fig. 1B). However, by 5' RACE assays, we found two fragments of different lengths, which when sequenced proved to result from differences in the length of 5'-UTRs of otherwise identical messages (Fig. 1A). When the sequences of these 5'-UTR variants were compared with the CytMe genomic sequence (GenBank accession no. AY033591), we found that they result from alternative transcription start sites and not from alternative 5' leader exons. The longer transcript begins 225 bases upstream of the shorter counterpart. The size of the longer message is apparently exaggerated to 2.4 kb in gel electrophoresis because of the extensive secondary structures in its 5' leader (see below). For simplicity we continue to refer to this mRNA by its size (2.4 kb) relative to size markers in electrophoresis. Of 20 independently cloned and sequenced mRNAs from F. trinervia, all were identical in sequence except for the length of 5' leader, consistent with the presence of only a single CytMe gene. The 5'-UTRs of F. trinervia CytMe transcripts include the eight bases that distinguish the F. pringlei CytMeB from the CytMeA transcripts, and they include an additional three bases not observed in the UTRs of other Me mRNAs.

The m-fold algorithm (version 3.1; Mathews et al., 1999; Zuker et al., 1999) predicts four different stem-loop structures made possible by the additional 225 bases in the 5' leader of the longer (2.4-kb) CytMe transcript. These structures include the entire 5'-UTR from eight bases upstream of the AUG to seven bases from the 5' end. It is possible that these stable secondary structures might influence mRNA stability, processing or translation efficiency (see "Discussion").

The Expression of CytMe Is Affected by Light Only in Leaves

To compare the influence of light on the expression of CytMe and ChlMe genes in C₃ and C₄ species, mature greenhouse-grown plants were dark-adapted for 5 d and then re-illuminated. In F. pringlei, a single 2.0-kb CytMe transcript accumulated in light-grown leaves, the level of which remained unchanged during dark treatment and re-illumination (Fig. 5A). However, dark adaptation produced an additional 2.4-kb CytMe transcript, the levels of which peaked at 8 h after re-illumination and then decreased. ChlMe transcripts could not be detected in these leaves.

View larger version (72K): [in this window] [in a new window]   Figure 5.   Effect of illumination on CytMe and ChlMe mRNA levels in F. pringlei and F. trinervia. A, Total RNA isolated from fully expanded leaves of plants grown in a greenhouse, dark-adapted for 5 d, and then returned to the greenhouse for 2 to 24 h. B, Total RNA isolated from roots of soil-grown plants (R) and roots of medium-grown plants (GR, green roots). GR1 and GR2 represent two different preparations. To normalize RNA loading, the blot was stripped and rehybridized with a ubiquitin probe (Ubi). Photographs of the 25S rRNA bands from the ethidium bromide-stained gels are also shown. Approximately 20 µg of total RNA was loaded per lane.

In F. trinervia leaves, two CytMe transcripts were present before the plants were shifted to darkness (Fig. 5A). The longer 2.4-kb transcript disappeared during dark adaptation and subsequent re-illumination, a pattern opposite to that observed in F. pringlei leaves. However, the level of the 2.0-kb transcript remained unaltered throughout the procedure. As expected for a C₄ species, ChlMe expression was positively regulated by light.

The effect of direct illumination on the accumulation of CytMe mRNA was also examined in roots of F. pringlei and F. trinervia, which green when exposed to direct light (Fig. 5B). Exposure to light had no effect on the expression of CytMe in roots of either species. In contrast, direct light exposure repressed the expression in roots of ChlMe genes in F. pringlei and induced expression in F. trinervia. Because illuminated roots were grown in medium with Murashige and Skoog salts and Suc rather than soil, it is possible that these patterns were influenced by Suc or other components of the medium.

CytMe Expression Is Influenced by Mechanical Injury

Because CytMe expression patterns have previously been shown to respond to wounding, possibly as a component of defense responses (Schaaf et al., 1995; Casati et al., 1999a; Drincovich et al., 2001), we compared CytMe and ChlMe mRNA levels in fully expanded leaves of the C₄ species F. trinervia after wounding treatments (Fig. 6). Fully expanded leaves of F. trinervia accumulate two CytMe transcripts of approximately 2.0 and 2.4 kb. Within 3 h of wounding, however, a third CytMe transcript of approximately 2.2 kb can be detected. The level of the 2.2-kb transcript increased with time after wounding for at least 7 h, whereas the level of the 2.4-kb transcripts decreased. This suggests that wounding induced a change in the start sites or processing of CytMe messages. In the same leaves, the level of ChlMe mRNA was unaffected by wounding.

View larger version (87K): [in this window] [in a new window]   Figure 6.   Effect of wounding on CytMe and ChlMe mRNA levels in fully expanded F. trinervia leaves. Leaves from the same node on a greenhouse-grown plant was mechanically wounded before samples were collected at the indicated times (see "Materials and Methods" for details). The experiment was repeated twice and similar results were obtained each time. To normalize RNA loading, the blot was stripped and rehybridized with a ubiquitin probe (Ubi). A photograph of the 25S rRNA bands from the ethidium bromide-stained gel is also shown. Approximately 20 µg of total RNA was loaded per lane.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Potential Roles of CytMe in C₃ and C₄ Species

CytMe mRNA species of 2.0, 2.2, and 2.4 kb are differentially present in C₃ and C₄ Flaveria spp. in patterns that suggests they correspond to roles for cytosolic ME in general metabolism, in wound responses, and in the control of cytoplasmic pH. In stems and roots of F. trinervia and in all organs of F. pringlei, only the 2.0-kb transcript accumulates. In leaves of both F. pringlei and F. trinervia, the 2.0-kb transcript increases with the extent of leaf maturation but is not influenced by light or wounding. The ubiquitous presence of the 2.0-kb transcript suggests that it is responsible for the majority of CYTME activity in both C₃ and C₄ plants. The 2.2- and 2.4-kb transcripts appear under more specific conditions.

Mechanical wounding in fully expanded F. trinervia leaves leads to accumulation of the 2.2-kb CytMe transcript at the expense of the 2.4-kb transcript. During injury, significant levels of reducing agents and carbon metabolites are needed to support cell wall building. Accumulation of the 2.2-kb CytMe transcript suggests that CYTME is involved in the supply of NADPH and pyruvate for this process. It is noteworthy that wound-induction of a cytosolic NADP-ME has been reported in bean (Phaseolus vulgaris), a C₃ plant (Schaaf et al., 1995).

The expression pattern of the 2.4-kb CytMe transcript indicates a correlation between this transcript and the ME required for controlling cytoplasmic pH in leaves. The phenomenon of illumination-induced cytoplasmic alkalization has been compared in the C₃ plant broad bean (Vicia faba) and the C₄ plant Amaranthus caudatus (Raghavendra et al., 1993; Yin et al., 1993). Whereas darkening led to rapid acidification in the C₃ leaf, alkalization continued in the darkened C₄ leaf. The observation that cytoplasmic pH can be controlled by balancing malate synthesis and degradation (Davies, 1973; Raven and Smith, 1974; Smith and Raven, 1979; Martinoia and Rentsch, 1994) provides a rationale for the opposite effects of illumination on the accumulation of the 2.4-kb CytMe transcript in the leaves of C₃ and C₄ Flaveria spp. (Fig. 5A). The appearance of the 2.4-kb CytMe transcript in F. pringlei (C₃) upon dark-adaptation suggests that CYTME is induced for malate degradation to offset acidification. By the same rationale, continued alkalization in the dark in F. trinervia (C₄) might repress CYTME, as evidenced by the decrease in the 2.4-kb transcript (Fig. 5A), and thereby regulate the pathway in the opposite direction.

Although this study provides no direct experimental evidence regarding why CytMe mRNA species of different lengths (2.0, 2.2, and 2.4 kb) are synthesized to execute roles in general metabolism, wounding responses, and control of cytoplasmic pH, it is tempting to speculate on the bases for this finding. It is evident that depletion of the malate pool will result if dark-adaptation-induced acidification is to be offset or if NADPH levels need to be increased subsequent to wounding. However, the essential requirement of a large malate pool for photosynthesis in the C₄ species F. trinervia likely imposes an acute need for regulatory mechanisms that will precisely control the flux of malate for other cellular requirements. Although the levels of ME need to be rapidly increased if malate is to be degraded, the ME levels needs to be decreased immediately if the malate levels reach a lower threshold. It is in this dual context of rapid inducibility and repression that the different transcripts produced from the single CytMe gene in Flaveria spp. might play a role. First, because the 2.0- and 2.4-kb mRNAs (and perhaps the 2.2-kb mRNA) appear to vary only in the length of 5'-UTR, it is plausible that these mRNA species possess sequences/structures that render them suitable for acting as molecular switches that respond to physiological stimuli. The four alternative stem-loop structures predicted in the 5'UTR of the 2.4-kb mRNA all have a free energy of approximately 50 kcal/mol. Although such stable structures are usually associated with decreased translational efficiency, it has recently become evident that such structures can serve as regulatory elements. "Sensory" mechanisms controlling translation have been reported (Cereghino et al., 1995; Nielsen et al., 1995; Luo and Sachs, 1996; Wells et al., 1998; Morita et al., 1999; Diwa et al., 2000; Ravnum and Andersson, 2001). The introduction of secondary structures in the 5'-UTRs raises the possibility of translational control through trans-acting factors, a feature already documented in mammalian cells and plant systems (Kozak, 1999; Morris and Geballe, 2000; Wang and Wessler, 2001). The differences in the length of the CytMe mRNA transcripts (i.e. 2.2 versus 2.4 kb) subsequent to wounding and dark adaptation might reflect differences in the trans-acting factors that influence the expression of CYTME under these conditions. Second, the presence of the 5'-UTRs might enhance mRNA stability (Cereghino et al., 1995; Arnold et al., 1998) and thereby assist in fulfilling the need for higher CYTME activity during sustained increases in levels of cytosolic malate or acidity. Lastly, the significant accumulation of the longer transcripts might indicate that the mature mRNA transcript is generated by processing of a longer precursor transcript. This processing event might be conditionally regulated and leads to an mRNA that is translated with a higher efficiency and thereby fulfill the requirement for higher CYTME levels during the induction phase.

It is striking that in Flaveria spp., the multiple copies of the CytMe genes found in C₃ species are reduced in C₃-C₄ and C₄ species to a single copy. This may reflect the need in these latter species for rapid cellular responses to changes in the pool of C₄ metabolites to prevent futile cycling. Such rapid changes might be difficult to achieve by shifts in transcription to up- and down-regulate distinct ME genes and more easily achieved by transcriptional, post-transcriptional, and translational controls on a single gene, in addition to regulation of the enzymatic activity.

Correspondence between NADP-ME Genes and Protein Isoforms in Flaveria Species

Three NADP-ME isoforms, of molecular masses 62, 64, and 72 kD, have been detected immunologically in all Flaveria spp. (Drincovich et al., 1998; Casati et al., 1999b). The abundance of the 62-kD isoform is correlated with C₄ photosynthetic capacity, suggesting that the 62-kD isoform is the C₄-specific CHLME encoded by ChlMe1. Consistent with this, ChlMe1 is highly transcribed specifically in the leaves of the C₄ species F. trinervia (Marshall et al., 1996; L. Lai, L. Wang, and T. Nelson, unpublished data). In the leaves of C₃ Flaveria spp. and non-photosynthetic organs of all Flaveria spp., the pool of NADP-ME was found almost exclusively to consist of the 72-kD isoform, similar to our finding with the CytMe mRNAs. Therefore, we suggest that the ubiquitous 72-kD isoform is encoded by the constitutively transcribed CytMe gene, because transcript and protein localization patterns show no correlation with photosynthesis. In contrast, the 64-kD isoform is found at higher levels in C₄-like and C₃-C₄ species (Drincovich et al., 1998) and is therefore likely to be encoded by ChlMe2; further biochemical studies are required to verify this premise. The levels and cellular localization of the 62- and 64-kD isoforms are similar in C₃-C₄ species (Drincovich et al., 1998; Casati et al., 1999b). It is likely that in the ancestral C₃ species, ChlMe1 and ChlMe2 were regulated in a similar fashion. When a chloroplastic NADP-ME activity was required early in C₄ evolution, as represented by Flaveria C₃-C₄ species, the expression of both genes might have been increased, followed by the divergence and distinct expression patterns of the genes exhibited in C₄ species.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Materials and Treatments

Multiple isolates of Flaveria spp. were provided by Dr. Scott Holaday (Texas Tech University, Lubbock), Dr. Harold Brown (University of Georgia, Athens), Drs. Maurice S. B. Ku and Gerald Edwards (Washington State University, Pullman), and Dr. Peter Westhoff (Heinrich-Heine-Universitat, Dusseldorf). Flaveria trinervia and Flaveria bidentis were grown from seeds, whereas all other Flaveria spp. were propagated from vegetative cuttings. Greenhouse-grown plants were used for light-shift and wounding experiments as well as for isolation of mRNA for the Flaveria pringlei leaf cDNA library. The greenhouse was maintained at an average temperature of 28°C during summer and 25°C during winter, with supplemental lighting to obtain a 16-h light/8-h dark cycle in the range of 700 to 1,000 µmol m² s¹. For all other experiments, plants were maintained in a controlled growth room at 28°C with a 16-h light/8-h dark cycle at 700 to 900 µmol m² s¹ supplied by a combination of white fluorescent and incandescent lights.

For northern-blot analysis of CytMe expression during leaf development, leaf length was used as an indicator of expansion stage in plants grown in a growth chamber. Leaves ranging in size from 1 cm to the maximum lengths of 13 cm (for F. pringlei) and 8 cm (for F. trinervia) were collected. Senescing leaves were avoided by collecting leaves only from the top five nodes of plants with at least 10 nodes.

In re-illumination experiments, only fully expanded leaves from greenhouse-grown plants at the 6- to 7-node stage were used, to eliminate the effects of developmental regulation. After being dark-adapted for 5 d, plants were re-illuminated in the greenhouse for up to 24 h and leaves were collected at the indicated times.

In wounding experiments, the youngest pairs of fully expanded leaves from three different mature F. trinervia plants (greenhouse-grown) were treated. Each leaf was severed along both sides of the midrib, yielding two halves of midrib-less blades that were still attached to the plant by the petiole. Each plant therefore yielded four leaf-halves, each of which was collected at 0 (control), 3, 5, and 7 h after treatment.

Oligonucleotides

The following oligonucleotides were used in the various parts of this study: VN-1, 5'-ACCATGATTTCCTTGAACTC-3'; VN-2, 5'-ACCATGATTTCCTTGAACTC-3'; FtrMe-3, 5'-GCCACCGGAGCATTCACATCC-3'; CytMe-1, 5'-TTGCGTCCTCGAGCCTCCGG-3'; CytMe-1A, 5'-CCAAAGTGTTTGCTTTATC-3'; CytMe-2, 5'-AAGCTTATTTTTGTTATTGAACT-3'; CytMe-3, 5'-CGTGCACTATGTATGAGTTG-3'; CytMe-4, 5'-AATATTTTGCATCAATTTCTTCT-3'; CytMe-5, 5'-GTGGTATGATGGAACTCG-3'; CytMe-6, 5'-GAACTCTTCAAGTAAATCATA-3'; CytMe-7, 5'-GGTGAAAAGGTCCTCGTG-3'; CytMe-8, 5'-TTAGAACAGATGGTTTGATG-3'; CytMe-9, 5'-GAGGCATGGCAGCCTT-3'; CytMe-10, 5'-TCACCAGATCAGTAGGGC-3'; Cy5R1, 5'-CCTCGGAGGCRCGGTCMTC-3'; Cy5R2, 5-CGCCACCGCCCACGGC-3; Cy5R5, 5'-GTGGATCCCTCAGCAAAGTG-3'; Trcytlong, 5'-GAGAGTAAAATTGGAATTTATATT-3'; Tanchor, 5'-GGATCCGTCGAGT(15)-3'; and Tadaptor, 5-GGATCCGTCGAGTTT-3'.

Isolation of CytMe cDNAs

Poly(A) RNA was isolated from fully expanded leaves of greenhouse-grown F. pringlei and was used in constructing a cDNA library with the ZAP-cDNA Synthesis Kit (Stratagene, La Jolla, CA). The library was screened with a 322-bp fragment encoding the chloroplast transit peptide and the first 45 amino acids of CHLME1. The latter region is very similar in CHLME and CYTME proteins. The 322-bp probe was generated by RT-PCR using the primers VN-1 and VN-2 and F. trinervia leaf RNA (B. McGonigle and T. Nelson, unpublished data). Successive rounds of screening were carried out by hybridization in 5× SSC, 5× Denhardt's, 0.1% (w/v) SDS, 0.1% (w/v) tetrasodium pyrophosphate (Na₄PPi), and 200 µg mL¹ salmon sperm DNA at 65°C. Subsequently, two 30-min washes at 65°C were performed using 1× SSC, 1% (w/v) SDS, and 0.1% (w/v) Na₄PPi as the first wash and 0.1× SSC, 1% (w/v) SDS, and 0.1% (w/v) Na₄PPi as the second wash. Sequence assembly and analysis were performed using GeneWorks (version 2.5.1, Intelligenetics, Inc., Mountain View, CA).

Labeling of Gene-Specific Fragments for DNA and RNA Gel-Blot Analyses

PCR was used to radiolabel fragments (Mertz and Rashtchian, 1994) with 35 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s. The CytMe-specific fragment was obtained using the F. pringlei CytMeA cDNA as template and the primers CytMe-3 and VN-2. This probe comprises the 5' portion of CytMeA cDNA, which has low homology to the corresponding regions in the ChlMe cDNAs (Fig. 1A). The ChlMe-specific fragment was obtained using the 322-bp fragment described above as template and the primers VN-1 and FtrMe-3. This probe contained the complete coding region for the plastidic transit peptide which is unique to ChlMe genes (Fig. 1A).

Estimation of Gene Copy Number

Ten to 15 µg of genomic DNA was digested with restriction enzymes, separated by electrophoresis in a 1% (w/v) agarose gel, and then transferred to Nytran Plus membrane (Schleicher & Schuell, Keene, NH). To ensure hybridization to genomic DNAs from all species used, low-stringency hybridization was carried out in 25% (w/v) formamide, 5× SSC, 5× Denhardt's, 1% (w/v) SDS, 0.05% (w/v) Na₄PPi, 250 µg mL¹ salmon sperm DNA, and 5% (w/v) dextran sulfate at 42°C. Subsequently, two 20-min washes at 42°C were performed using 2× SSC, 0.1% (w/v) SDS and 1× SSC, 0.1% (w/v) SDS as the first and second wash solutions, respectively.

Measurement of CytMe and ChlMe mRNA Levels

Total RNA was isolated using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. Approximately 20 µg of total RNA was separated by formaldehyde agarose gel electrophoresis and transferred to Nytran Plus membrane (Schleicher & Schuell, Keene, NH). High-stringency hybridization was carried out in 50% (w/v) formamide, 5× SSPE, 5× Denhardt's, 0.5% (w/v) SDS, 25 mM EDTA, 200 µg mL¹ salmon sperm DNA, and 5% (w/v) dextran sulfate at 42°C. Subsequently, two 20-min washes were carried out at 42°C with 2× SSC, 0.1% (w/v) SDS. Blots were analyzed using the Fujix BAS-2000 phosphorimager (Fuji, Tokyo).

Ubiquitin mRNA levels were analyzed to normalize for differences in RNA loading. Two ubiquitin probes were used. The first probe, the insert of pSKUB1 (Christensen and Quail, 1989), was randomly labeled and used with the high-stringency hybridization method. The second ubiquitin probe was a 5' end-labeled oligonucleotide UBQRep (5'-CTCCTTCTGGATGTTGTAGT-3'), which is complementary to the repeat sequences highly conserved among all known plant ubiquitin mRNAs (Xia and Mahon, 1998). The oligonucleotide probe was hybridized in 0.6 M NaCl, 0.18 M Na₂HPO₄, 6 nM EDTA, pH 6.8, 0.75% (w/v) N-lauroyl-sarcosine, 10% (w/v) dextran sulfate, and 0.5 mg mL¹ heparin (Dellaporta and Moreno, 1994) at 42°C and washed twice in 6× SSC, 0.1% (w/v) SDS for 10 min each at 37°C. Both ubiquitin probes detected three mRNA species of sizes 0.8, 1.6, and 2.1 kb in F. pringlei RNA samples, as reported for maize (Christensen and Quail, 1989). In F. trinervia RNA samples, only the 0.8- and 2.1-kb mRNAs were detected.

Determination of mRNA Size Variants

Total RNA from young F. trinervia leaves (up to 4 cm long) was used for all of the following experiments. For detection of alternative splice events, the first-strand cDNA was obtained using the RETROscript kit (Ambion, Austin, TX) and the oligo(dT) primer. Regions of the cDNA suspected to contain introns were amplified using PCR for 35 cycles at 94°C for 45 s, 50°C for 45 s, and 72°C for 1 min. Positive and negative controls were included in each run and each region was tested at least twice. The primers used were CytMe-2 through CytMe-10.

3' RACE was carried out using the primer Tanchor and Superscript II reverse transcriptase (Life Technologies). Glass Max spin columns (Life Technologies) were used to rid the product of extra primers. PCR reactions included the CytMe-specific primer CytMe-1 (194 bp upstream of the major poly[A] site) and the primer Tadaptor for the first round and CytMe-1A and Tadaptor for the second round.

5' RACE was carried out using the 5' RACE kit Version 2.0 (Life Technologies) and the CytMe-specific primer CytMe-4 (291 bp downstream of the start codon), followed by two rounds of PCR amplification with the nested primers Cy5R5 and Cy5R1. Potential RNA secondary structure within the 5'-UTR was identified using the RNA m-fold algorithm (version 3.1) at http://bioinfo.math.rpi.edu/~mfold/, maintained by M. Zuker (Rensselaer Polytechnic Institute). To determine the origin of the 5'-UTR extension in the longer CytMe transcript, its sequence was compared with the genomic sequence. The genomic F. trinervia DNA was amplified first using the primers Cy5R1 and Trcytlong. The products from this reaction were re-amplified using Trcytlong and Cy5R2 as the primers.

PCR products from the 5' RACE reactions, genomic amplification, and some of the reactions designed to detect alternate splicing were cloned (TOPO TA kit, Invitrogen, Carlsbad, CA) and sequenced (Keck Sequencing Facility, Yale University).