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Genetics |
Virginia Tech, Blacksburg, Virginia 24061 USA
Received for publication August 27, 2006. Accepted for publication June 15, 2007.
ABSTRACT
Strong phylogenetic signal from matK has rendered it an invaluable gene in plant systematic and evolutionary studies at various evolutionary depths. Further, matK is proposed as the only chloroplast-encoded group II intron maturase, thus implicating MATK in chloroplast posttranscriptional processing. For a protein-coding gene, matK has an unusual evolutionary mode and tempo, including relatively high substitution rates at both the nucleotide and amino acids levels. These evolutionary features have raised questions about matK function. In the current study, we examined matK RNA and protein from representative land plant species to provide insight into functional aspects of this unusual gene. We report the first evidence of a transcript for matK separate from the trnK precursor and demonstrate that a full-length MATK protein exists in five angiosperm species. We also show that matK RNA and protein levels are regulated by light and developmental stage, suggesting functional roles for this putative maturase. Specifically, matK expression increased after etiolation and decreased at 4 weeks after germination. This work provides evidence for the expression of the only putative chloroplast-encoded group II intron maturase and insight into regulation mechanisms relating to plant development and, indirectly, to photosynthesis.
Key Words: developmental stage • etiolation • expression • matK • maturase • systematics
In plant systematics, matK has recently emerged as an invaluable gene because of its high phylogenetic signal compared with other genes used in this field (Müller et al., 2006
). The 1500 bp matK gene is nested in the group II intron between the 5' and 3' exons of trnK in the large single copy region of the chloroplast genome of most green plants (e.g., Sugita et al., 1985
; Steane, 2005
; Daniell et al., 2006
; Turmel et al., 2006
). Phylogenetic analysis of a data set composed of matK, rbcL, and trnT-F sequences from basal angiosperms demonstrated that matK contributes more parsimony informative characters and significantly more phylogenetic structure on average per parsimony-informative site than the highly conserved chloroplast gene rbcL (Müller et al., 2006
). Sequence information from matK alone has generated phylogenies as robust as those constructed from data sets comprised of 2–11other genes combined (see Hilu et al., 2003
). Further, the molecular information generated from matK has been used to resolve phylogenetic relationships from shallow to deep taxonomic levels (Johnson and Soltis, 1994
; Hayashi and Kawano, 2000
; Hilu et al., 2003
; Cameron, 2005
).
The matK gene stands out among plastid genes used in plant systematics in its distinct mode and tempo of evolution. The rate of substitution in matK is three times higher at the nucleotide level and is six times higher at the amino acid level than that of rbcL (Johnson and Soltis, 1994
; Olmstead and Palmer, 1994
), denoting it as a fast or rapidly evolving gene (Soltis and Soltis, 2004
). The accelerated rate of amino acid substitution in matK is due to almost even distribution of substitution rates among the three codon positions compared with most protein-coding genes where the rates are skewed toward the third codon position. For example, substitution rates for the three codon positions of matK were 62%, 57%, and 66%, respectively, when comparing angiosperms at the ordinal level (Hilu et al., 2003
), and 32%, 28%, and 39%, respectively, in Orchidaceae (Whitten et al., 2000
). Insertions and deletions (indels) are frequent in matK, though these indels primarily occur in multiples of three, maintaining the reading frame. Further, the transition/transversion ratio in the gene approaches unity in sequences examined at both deep (order) and shallow (subfamily) taxonomic levels (Liang and Hilu, 1996
; Hilu et al., 2003
). The rapid rate of substitution, along with the rare presence of frameshift indels and a few cases of premature stop codons, prompted some researchers to suggest that matK may not be functional in some plants (Kores et al., 2000
; Whitten et al., 2000
; Kugita et al., 2003
; Hidalgo et al., 2004
; Jankowiak et al., 2004
).
Sequence features alone, however, provide inconclusive evidence for lack of function. RNA editing mechanisms previously reported in matK (Vogel et al., 1997
; Tillich et al., 2005
) may correct the reading frame in species with frameshift indels and premature stop codons and restore the codon identities needed to form the proper amino acids for function. Further, genome studies of the holoparasite Epifagus virginiana (Ems et al., 1995
) and Adiantum capillus-veneris (Wolf et al., 2003
) support that matK has a function in the plant. Epifagus virginiana, which has lost more than 60% of its chloroplast genome including trnK, retained matK (Ems et al., 1995
). Similarly, a large rearrangement in the chloroplast genome of Adiantum resulted in the loss of trnK but retention of matK (Wolf et al., 2003
). The maintenance of the matK gene in both species after loss of several other genes suggests that this gene is expressed and serves an essential and irreplaceable function in the plant.
In addition to the importance of matK in plant phylogenetics, it is also the only putative group II intron maturase encoded in the chloroplast genome (Neuhaus and Link, 1987
). Maturases are enzymes that catalyze nonautocatalytic intron removal from premature RNAs. Maturases generally contain three domains: a reverse-transcriptase (RT) domain, domain X (the proposed maturase functional domain), and a zinc-finger-like domain (Mohr et al., 1993
). The 3' region of matK has homology to the domain X of mitochondrial group II intron maturases (Neuhaus and Link, 1987
). This region of matK also lacks indels (Hilu and Liang, 1997
), indicating evolutionary constraint and conservation of function. Among higher plants, matK is the only plastid gene containing this putative maturase domain (Neuhaus and Link, 1987
). The gene, however contains only remnants of the RT domain and no evidence of the zinc-finger-like domain found in other maturases (Mohr et al., 1993
). Although at present there is no direct evidence for the maturase function of MATK, indirect evidence from the white barley ribosomal mutant albostrians supports this proposed function. Vogel et al. (1997
, 1999
) demonstrated that the albostrians mutant, which lacks all chloroplast ribosome activity and, subsequently, all chloroplast proteins including MATK, lacks group II intron excision from certain premature RNAs. Based on this and other studies, researchers have proposed that the following RNA transcripts require MATK for intron excision: trnK, trnA, trnI, rps12, rpl2, and atpF (Ems et al., 1995
; Jenkins et al., 1997
; Vogel et al., 1999
). The tRNA or protein products from these genes are required for normal chloroplast function including photosynthesis. The above considerations suggest that MATK has an essential function in the chloroplast as a posttranscription splicing factor.
Previous studies examining matK RNA and protein have yielded conflicting results concerning whether this gene is expressed. While several studies have observed a matK/trnK transcript (Kanno and Hirai, 1993
; Vogel et al., 1997
; Kugita et al., 2003
; Nakamura et al., 2003
; Wolf et al., 2004
), the majority of these studies did not determine whether this transcript proceeded to translation of MATK protein or was merely a premature unspliced trnK tRNA that happened to include the matK open reading frame (ORF). Only one study, which used Hordeum vulgare L. (barley, Poaceae) investigated whether a monocistronic matK transcript existed separate from trnK (Vogel et al., 1997
). The results from this study indicated that a matK transcript may not exist separately from a trnK unspliced precursor. This finding suggests that matK is not translated into protein, or is translated from a transcript present in very low levels (Vogel et al., 1997
), or cannot be easily distinguished from the trnK precursor. Thus, the question of whether matK is an expressed gene with its own transcript separate from premature trnK remains largely unanswered.
Three studies have presented evidence for the existence of a MATK protein. Two of these studies, du Jardin et al. (1994)
using Solanum tuberosum L. (potato, Solanaceae) and Liere and Link (1995)
using Sinapis alba L. (Brassicaceae), identified a MATK protein of much lower molecular mass than expected for the full-length ORF. These results indicate that MATK is truncated. In contrast, a later study using barley identified a protein product close to the expected molecular mass for full-length MATK (Vogel et al., 1999
), confirming that matK is expressed and that the protein is not truncated in this plant species. Consequently, further studies at the RNA and protein level are needed to examine expression and function of this gene.
The objectives of the current study were two fold: (1) to determine the presence of transcription and translation products for matK and assess the relationship of matK to the expression of trnK, and (2) to examine possible functions for this putative maturase. To address these objectives, we first determined the number and size of transcripts generated from the matK/trnK gene region in two model species Oryza sativa L. (rice, Poaceae) and potato. Second, we used several methods to examine whether matK is transcribed independently from trnK. Third, we investigated whether a protein product of the expected size for full-length MATK is produced in several angiosperm species. Fourth, we investigated possible functions for MATK in rice by assessing RNA and protein levels in response to etiolation and during postgermination development. This work provides the first detailed examination of the expression of this potentially essential chloroplast maturase.
MATERIALS AND METHODS
Plant material for RNA analyses
Two angiosperms models, one monocot (rice) and one eudicot (potato), were used in this study to avoid possible bias to a single angiosperm species. The choice of using potato vs. a more common eudicot model such as Arabidopsis for our expression studies was based on a previous investigation of matK expression and function, which used potato as the model (du Jardin et al., 1994
). In addition to these two models, 14 species representing the four major land plant lineages (bryophytes, monilophytes, gymnosperms, and angiosperms) were used to survey matK RNA (Appendix 1). Species chosen within angiosperms represented basal and late-diverging lineages (Table 1). Taxa sampling in monocots was extended to represent sections of their phylogeny (Hilu et al., 2003
) from early diverging Alismatales (Sagitaria latifolia Willd., Alismataceae), to later diverging Poales (rice and Zea mays L., Poaceae) and Asparagales (Spathoglottis plicata Blume, Orchidaceae). The orchid Spathoglottis was specifically chosen to investigate comments that matK is a pseudogene in the chloroplast genome of this plant (Freudenstein et al., 2004
). By including sample taxa of basal-, mid-, and late-diverging lineages, our survey accounts for differences in angiosperms due to evolutionary distance and presents a broad representation across this plant group. Plants used in the current work were obtained from three sources: seed stock at the Virginia Tech Biology greenhouse, field collections, or potted plants purchased from private companies (for details of plant collections see Appendix 1). Both young and adult leaf material were collected for each plant, placed in zip-seal freezer bags, and stored at –80°C. Herbarium vouchers are deposited in the herbarium at Virginia Tech (VPI), Technische Universität Dresden (DR), or as part of the personal collection of Dietmar Quandt (Quandt), Technische Universität Dresden.
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). The rice matK ORF probe consisted of 876 base pairs (bp) that included 250 bp of the conserved maturase domain X (Mohr et al., 1993
) and was PCR-amplified using primers W and 9R (for information on primers, see Appendix S1 in Supplemental Data accompanying the online version of this article) (Fig. 1A). The potato matK ORF probe consisted of a 333-bp product of the matK N-terminal region and was amplified using the primers corematK1 and corematK2. Specificity of these two probes to matK was tested by Southern blot (see following section on Southern blot). Two rice trnK probes were designed to exclusively hybridize to the 5' or 3' exon of trnK (Fig. 1A). The primers 5extrnKF and trnK5exR were used to amplify a sequence segment starting from 132 bp upstream of the beginning of the 5' trnK exon to its end to generate the 5' trnK exon probe. Primers trnK3exF and trnK3exR were used to amplify a sequence section starting from 71 bp upstream of the beginning of the trnK 3' exon to the end of that exon to generate the 3' trnK exon probe. Dig-labeled probe synthesis was performed according to supplier instructions (Roche, Indianapolis, Indiana, USA).
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). The chloroplast genes matK and rbcL and the mitochondrial gene mat-r were amplified from rice genomic DNA using primer combinations W/9R (matK), rbcLOsF/rbcLOsR (rbcL), and matRF/matRrevdX (mat-r). Amplification product of mat-r included 186 bp of domain X. Amplified products were separated on a 1.5% agarose gel, transferred to a nylon membrane by capillary blotting in Church buffer (Church and Gilbert, 1984
), and hybridized with the matK ORF probes from rice and potato at low (50°C) and high (65°C) stringency. Probe signal was detected with anti-Dig and the chemiluminescent substrate CDP-Star (Roche, Indianapolis, Indiana, USA) followed by exposure on film.
RNA isolation and Northern hybridization analyses
Total RNA was isolated by grinding tissue under liquid nitrogen followed by phenol/chloroform/LiCl extraction and ethanol precipitation (Altenbach and Howell, 1981
). RNA was separated on a 1% formaldehyde gel according to Gerard and Miller (1986)
. RNA was transferred to a nylon membrane via capillary blotting in Church buffer and hybridized with the Dig-labeled matK ORF probes (described in the previous sections) at both low and high stringency or with the Dig-labeled trnK 5' and 3' exon probes at high stringency. Because RNA editing of matK has been observed previously (Vogel et al., 1997
; Wolf et al., 2004
), a low stringency hybridization was performed to ensure binding of matK ORF DNA probes to possible RNA-edited transcripts of this gene, which may vary slightly in sequence. Transcripts on membranes were visualized by incubation with CDP-Star followed by exposure on film.
3' RACE
Total RNA was isolated as described previously from rice tissue and stored at –80°C. RNA was DNase-treated using RQ1 RNase-free DNase (Promega, Madison, Wisconsin, USA) with the following modifications to the manufacturer's protocol: 1 µL of RNase inhibitor (40 u/µL, Invitrogen, Carlsbad, California, USA) was added along with 6.7 µL of 5x reverse transcriptase buffer (Invitrogen), 4.3 µL of DNase (1 u/µL), and up to 10 µg of total RNA for a 20-µL reaction. 3' RACE was performed using the TaKaRa 3'-Full RACE Core Set (TAKARA BIO, Otsu, Shiga, Japan) according to the manufacturer's directions with the exception of using trnK3exR as the reverse primer instead of oligo dT in the RT reaction for mature trnK cDNA. The primer combinations W/trnK3exR, W/9R, and 3914/trnK3exR were used in a PCR reaction with cDNA generated from 3' RACE to amplify transcripts of the matK ORF to the end of the 3' trnK exon, the matK ORF alone, and the mature transcript of trnK, respectively. A negative control lacking reverse transcriptase (no-RT control) was included in the synthesis of cDNA using 3' RACE.
Reverse transcription (RT)-PCR
Prior to RT-PCR, all RNA samples were amplified in a PCR reaction that included RNase to check for DNA contamination. RNA samples contaminated with DNA were treated with DNAse as described previously. First strand synthesis was performed according to Shirley and Hwang (1995)
with the following modifications: 1 µL Superscript II reverse transcriptase (200 u/µL, Invitrogen) was used instead of Moloney Murine Leukemia Virus reverse-transcriptase and an oligo dT(15) primer was used for the reverse primer. The reaction was incubated at 37°C for 30 min for first strand synthesis.
The cDNA was amplified using gene-specific primers designed to anneal to the 5' region of matK distant from the conserved domain X. Some primers were species- or family-specific; however, degenerate primers that could span large groups of plants and remain specific to matK were made where possible (for information on primers, see Appendix S1 in Supplemental Data accompanying the online version of this article). A negative control containing RNA not reverse transcribed (no-RT) and RNase was co-amplified with cDNA, confirming that all PCR products from RT-PCR were the result of amplifying cDNA and not genomic DNA. All PCR products were separated on 1.5% agarose gels.
Sequencing
Amplified products from all PCR reactions, including generated probes and 3' RACE products, were gel-extracted using the Qiagen Qiaquick gel extraction kit (Qiagen, Valancia, California, USA) and sequenced to confirm their gene identity. Direct sequencing was performed using the BigDye Terminator Sequencing Kit (ABI, Framingham, Massachusetts, USA) with the same primers used in the initial reaction. Annealing temperature of the sequencing reaction varied depending on the primer used. Products of the sequencing reaction were separated through capillary gel electrophoresis at the Virginia Bioinformatics Core Laboratory Facility (Blacksburg, Virginia, USA).
Plant material for protein analyses
Nine plant species (eight angiosperms and one monilophyte) were used to determine whether matK protein was expressed in various plant species. Because the antigen used to generate the MATK antibody utilized in this study was from rice, sample taxa were predominantly limited to monocots in order to increase the likelihood of antibody binding. Sample taxa included three species of the Orchidaceae to address previous assertions that matK might not be functional in some members of this plant family (Kores et al., 2000
; Whitten et al., 2000
; Goldman et al., 2001
). The eudicot Arabdiopsis thaliana L. (Heynh.) (Brassicaceae) was used to test the threshold of homology required for antibody binding to protein from more evolutionary diverged angiosperms. Protein extract from the monilophyte Psilotum nudum L. (Psilotaceae) was used as a negative control. Although P. nudum may express MATK protein, the corresponding region in this fern to the peptide sequence used to produce our rice MATK antibody has a four-amino acid deletion (Fig. 1B) and low identity (13%) to the rice antigenic peptide sequence, rendering the cross-immunological reaction very unlikely. Plant material was obtained as described for RNA analyses (see Appendix 1). Leaf material was collected for each plant, placed in zip-seal freezer bags, and stored at –80°C. Herbarium vouchers are located at VPI. Vouchers are not available for specimens obtained from the University of Mary Washington.
MATK antibody design and testing
An antibody was produced against the 15-amino-acid rice MATK peptide sequence CPEEEKEIPKFQNLRS and synthesized by Cocalico Biologics (Reamstown, Pennsylvania, USA). The choice of peptide region for generating the MATK antibody was based on two criteria: (1) specificity to MATK and (2) applicability to a broad range of angiosperm species. To increase specificity of this antibody against MATK and not other maturases that may contain domain X, we chose the peptide sequence from the N-terminal region of MATK. This antigen sequence was demonstrated to be specific to MATK by blastp search in GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) using default settings, Viridiplantae as the organism group, and an inclusion threshold of 0.005. The 15-amino-acid peptide CPEEEKEIPKFQNLRS was also found to be fairly well-conserved across angiosperms by amino acid alignment (Fig. 1B). Comparison of this peptide sequence with the same region in other plant genera revealed almost 100% homology in the closely related grasses Avena sativa L. and Saccharum officinarum L. (Poaceae), 80% in Alisma canaliculatum A. Braun & Bouché (Alismataceae), 67% in Spathoglottis plicata Blume (Orchidaceae), 73% in Malleola baliensis J.J. Sm. and Holcoglossum tsii T. Yukawa (Orchidaceae), 40% in the eudicot A. thaliana, and 13% in the fern P. nudum (Fig. 1B). The specificity of this region to MATK, as determined by blastp and the conserved nature of this peptide across angiosperms, made this peptide region a strong candidate for the generation of a highly specific MATK antibody that could be used against protein extract from a variety of angiosperm taxa.
Antibody production in rabbits was carried out by Cocalico Biologicals. MATK antibody from the first bleed was purified by nitrocellulose absorption (A. Esen, Virginia Tech, personal communication) because of low antibody titer compared to background signal. Purification was not necessary for subsequent bleeds because of a much higher antibody titer than in the first bleed. Binding specificity of this antibody to MATK protein was tested by competition assay utilizing synthesized peptide antigen as the competing agent. Competition of antibody binding was demonstrated using 50 µg rice crude protein extract and 30 µg synthesized antigen resolved on a 7.5% SDS-PAGE gel. Resolved protein was then transferred to nitrocellulose and incubated with either unpurified rabbit MATK antibody (third bleed diluted 1 : 3000 in phosphate-buffered saline plus 0.1% Tween-20 [PBS-T] plus 3% bovine serum albumin [BSA]) or a mixture of this antibody plus synthesized antigen as the primary antibody. The MATK antibody and synthesized antigen mixture was incubated at room temperature for 1 h followed by 4°C overnight prior to being added to the membrane for the competition assay.
Protein extraction and Western blotting
Leaf or whole plant tissue was ground under liquid nitrogen and total protein extracted by the addition of Laemmli SDS sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and 5% ß-mercaptoethanol) plus 1 mM of the protease inhibitor phenylmethanesulfonyl fluoride (PMSF) followed by boiling at 95°C for 15 min. Denatured protein was centrifuged twice at 15 000 x g for 10 min each time, and the supernatant was retained after each spin. The final supernatant was stored at –20°C as crude protein extract. Protein concentration was determined by Bradford assay according to Jones et al. (1989)
using BSA as the standard. Fifty or 75 µg of crude protein extract from each plant species was fractionated by 7.5% SDS-PAGE and transferred onto nitrocellulose membrane. Equal loading of protein and transfer efficiency were confirmed by Ponceau S staining. For Western blotting, membranes were incubated with either nitrocellulose-absorption purified rabbit MATK antibody or pre-immune rabbit sera (diluted 1 : 50 or 1 : 300 in PBS-T) for the primary antibody. Horse radish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology, Danvers, Massachusetts, USA, diluted 1 : 2000 in 5% nonfat dry milk/PBST) was used as the secondary antibody for detection of immunoreactive protein. Bound antibody was detected using the ECL peroxidase/luminol system (Amersham Biosciences, Piscataway, New Jersey, USA) or West Pico chemiluminescent detection system (Pierce Biotechnology, Rockford, Illinois, USA).
Etiolation
Rice seed stock stored at 4°C was planted in vermiculite at the Virginia Tech Biology greenhouse and placed in two principal treatments: (1) under uniform light with a 10/14 day/night photoperiod in the greenhouse (the control) or (2) in the dark for 2 wk, hereafter referred to as either dark treatment or 0 h, reflecting the lack of light exposure. Both control and dark-treatment plants were uniformly watered. Dark-treatment plants remained in the dark while water was applied. Control plants from the greenhouse were harvested after 2 wk of growth. At 2 wk postgermination, a subset of plants from the dark treatment was harvested in a dark room. Remaining rice plants from the dark treatment were placed in a Percival growth chamber (Percival Scientific, Perry, Iowa, USA) and exposed to light for up to 24 h. Whole plant tissue was collected in two sets from these light-exposed plants: (1) after 4 h of light exposure and (2) after 24 h of light exposure. All tissue was placed in zip-seal freezer bags, frozen immediately in liquid nitrogen, and stored at –80°C. RNA and protein were isolated as described earlier. Transcript levels for matK/trnK and protein from each treatment were evaluated by Northern hybridization or Western blot (MATK antibody diluted 1 : 50 in PBS-T), respectively, as described earlier.
Developmental stage
Rice and potato plants were grown from seed stock and tubers, respectively, in the Virginia Tech Biology greenhouse under uniform light and water conditions. Plant leaves were collected at four times (2, 4, 6, and 8 wk postgermination), placed in zip-seal freezer bags, and stored at –80°C. RNA was isolated from both rice and potato, resolved on formaldehyde gels, and transferred to nylon membranes by the Northern blot method as described earlier. Membranes were hybridized with rice and potato matK ORF-Dig-labeled probes and detected by chemiluminescence using CDP-Star. Crude protein extract was isolated from rice leaves collected 2, 4, 6, and 8 wk postgermination, and analyzed by Western blot (unpurified MATK antibody diluted 1 : 300 in PBS-T) as described earlier.
RESULTS
matK RNA and protein
Hybridization of probes specific to the matK ORF to total RNA from rice and potato identified two predominant transcripts of 2.6 and 2.9 kilobases (kb) (Fig. 2A). Three smaller, less prominent, transcripts of approximately 1.8, 1.5, and 0.9 kb were also observed in both species (Fig. 2A). It is likely that these smaller transcripts represent splicing intermediates of the trnK/matK transcript. Transcripts of these same sizes were also identified in total rice RNA when using probes specific to the 5' and 3' trnK exons (Fig. 2B). In addition, the 5' trnK exon probe hybridized to a transcript of less than 0.5 kb, while the 3' trnK exon probe hybridized to a transcript of approximately 5.7 kb (Fig. 2B). A monocistronic matK transcript lacking the trnK exons was not found by Northern hybridization.
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RT-PCR using 3' RACE amplified an 876-bp product for matK, a 1377-bp product for part of the matK ORF to the end of the 3' trnK exon, and a 120-bp product for mature trnK (Fig. 3). The 3' RACE products were sequenced and confirmed as matK, matK with the 3' trnK exon, and mature trnK transcripts, respectively. The 120-bp size of the mature trnK transcript from 3' RACE corresponded well to the low molecular mass (<0.5 kb) transcript identified using the Dig-labeled 5' trnK exon probe in Northern hybridization. A no-RT control for the 3' RACE reaction using primers specific to the matK ORF (Fig. 3A) did not result in detectable product. These 3' RACE results confirm Northern blot data that a transcript containing both matK and the 3' exon of trnK does exist and that the mature trnK tRNA is approximately 120 bp in size and corresponds to a transcript band less than 0.5 kb on Northern blots.
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55-kDa protein was identified from rice protein extracts in Western blot experiments using an antibody targeted against rice MATK (Fig. 4A). This immunoreactive protein is close to the expected size (61 kDa) of the rice full-length MATK protein. Four additional protein bands of approximately 40, 30, 25, and 20 kDa were also identified (Fig. 4A). The 55-, 30-, and 20-kDa bands were not found when pre-immune serum was used on these same membranes (Fig. 4A). The 20-kDa band was not consistently observed with immunoblotting (data not shown). Competition experiments using an excess of synthesized antigen demonstrated successful competition of the MATK antibody to binding with the three protein bands of 55, 30, and 20 kDa (Fig. 4B), although less so for the 30-kDa band. Therefore, these immunoreactive protein signals are not the result of contaminating antibodies in the antipeptide serum but are specific to the MATK antibody. The 30- and 20-kDa immunoreactive bands are most likely products of MATK protein proteolysis. The 40- and 25-kDa bands were observed when blots were incubated with both MATK antibody and pre-immune serum (Fig. 4A) and are, therefore, considered the result of background pre-immune contaminating antibodies. These same background bands were not found on Western blots when using the third bleed of antibody serum, which contains a much higher titer of the MATK antibody (Fig. 4B).
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55-kDa immunoreactive protein decreased by about half in dark-treatment rice plants compared to the control. Levels of this protein increased slightly when etiolated plants were exposed to light (Fig. 5B). These results of protein levels for the 55-kDa immunoreactive protein parallel those of the 2.6- and 2.9-kb matK RNA transcripts and demonstrate light-induced expression or regulation for these RNA transcripts and the 55-kDa immunoreactive protein.
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55-kDa immunoreactive protein at 4 wk postgermination compared to the other time points (2, 6, and 8 wk postgermination) in rice (Fig. 5C). The decrease in the 55-kDa protein level corresponded to the decrease in RNA levels observed for the 2.6- and 2.9-kb matK/trnK RNA transcripts at 4 wk postgermination (Fig. 5C). These results indicate a relationship between plant developmental stage and expression of these RNA transcripts and the 55-kDa immunoreactive protein.
Survey of matK RNA and protein across land plants
RT-PCR on isolated DNA-free RNA produced a single predominant matK PCR product from 11 plant species representing three major plant lineages: bryophytes, monilophytes, and angiosperms (Table 1). The PCR products ranged from 333 bp to 876 bp, depending on the particular primer pair utilized for amplification (Table 1). All RT-PCR products resulting from this study were sequenced and confirmed as matK, with the exception of Adiantum hispidulum Swartz (Pteridaceae), in which low amplification prevented adequate sequencing. Amplification of no-RT controls did not result in PCR product, confirming that the RNA used for RT-PCR was devoid of genomic DNA contamination and that RT-PCR products were the result of cDNA amplification (Fig. 6A).
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).
A survey for expressed MATK protein in nine land plants resulted in the identification of MATK antibody-immunoreactive protein bands in six angiosperms (O. sativa, A. sativa, S. officinarum, Sagitarria latifolia Willd. [Alismataceae], Spathoglottis gracilis Rolfe ex Hook.f. [Orchidaceae], and A. thaliana) (Fig. 6B). The immunoreactive protein from five of these angiosperms was similar to the predicted size of the full-length MATK from each of these species (predicted/observed in kDa: 60/55 in O. sativa, 61/55 in A. sativa, 60/55 in S. latifolia, 62/60 in S. officinarum, and 60/55 in A. thaliana, Fig. 6). Protein bands of these same molecular masses were absent when immunoblots were incubated with pre-immune serum (Fig. 6). One additional anti-MATK immunospecific protein band of 20 kDa was seen on immunoblots with A. thaliana protein extract (Fig. 6). This additional band is most likely a product of MATK proteolysis. A unique MATK antibody-immunoreactive protein of 23 kDa was observed in S. gracilis (Fig. 6). No bands specific to the MATK antibody were found in extracts from the orchids M. ligulata and H. kimballianum (data not shown), or the fern P. nudum (Fig. 6B).
DISCUSSION
On the basis of the proposed substrates of MATK maturase activity, this putative enzyme critically impacts all chloroplast function including photosynthesis. In this study, we investigated whether matK is expressed and functional. Our results demonstrate that matK RNA and protein exist in several plant species. Further, we demonstrated that expression of matK is influenced by light and development stage, suggesting possible functions for this important maturase.
Independent transcripts for matK from the trnK precursor
Identification of the same 2.6- and 2.9-kb predominant transcripts by probes specific to the matK ORF and the 5' and 3' trnK exons (Fig. 2B) suggests that matK and trnK are transcribed as one dicistronic transcript. Vogel et al. (1997)
found similar results using RNA from barley and a probe specific to the 5' trnK exon. They identified two predominant transcripts of approximately 2.6 and 2.9 kb for the matK/trnK gene region and no evidence for a monocistronic matK transcript. Because it is unlikely that the same RNA transcript would form the mature trnK tRNA and at the same time proceed to translation for MATK protein, we hypothesize that the 2.6- and 2.9-kb transcripts represent mRNA transcripts dedicated to the formation of the MATK protein and are separate entities from the unspliced trnK precursor. While the trnK exons are present in this mRNA transcript, these exons act as the 5' and 3' untranslated regions (UTRs) of the mRNA and will therefore not form the mature trnK tRNA. We believe that the unspliced trnK precursor is very rapidly processed to produce the mature trnK tRNA-lysine and is not observable on Northern blots.
Several lines of evidence support our hypothesis that the 2.6- and 2.9-kb transcripts are dedicated to forming the MATK protein and are not unspliced trnK precursors. First, RNA levels for the 2.6-kb and 2.9-kb transcripts were light induced, while RNA levels of mature trnK tRNA remained constant (Fig. 5A, B). Thus, light is impacting expression of the high molecular mass transcripts differently than expression of the mature trnK tRNA, suggesting independent regulation of these transcripts. Second, levels of the 2.6- and 2.9-kb transcripts paralleled the expression of a 55-kDa protein immunoreactive to the MATK antibody in both etiolation and developmental stage experiments (Fig. 5). It appears, therefore, that these high molecular mass transcripts, which we have already determined by Northern hybridization to contain the matK ORF, are translated into a protein product that corresponds to the expected molecular mass of full-length MATK. Finally, RT-PCR experiments revealed that a matK transcript is present in the leptosporangiate fern Adiantum hispidulum (Fig. 6A). Similar results were also observed for A. capillus-veneris (Wolf et al., 2004
). Adiantum, unlike other plants used in this study, has a large rearrangement in its chloroplast genome in the trnK intron, resulting in the loss of trnK but retention of matK (Wolf et al., 2003
). The matK transcript identified in Adiantum by our study and by Wolf et al. (2004)
, therefore, could not possibly be the result of an unspliced trnK precursor transcript. Collectively, these results strongly support our hypothesis that the 2.6- and 2.9-kb predominant transcripts are dedicated matK mRNA transcripts and not the unspliced trnK precursor.
Rice MATK protein
We conclude that the 55-kDa immunoreactive band observed on Western blots containing rice crude protein extract is a full-length MATK protein (Fig. 4A). The rice MATK protein sequence (GenBank accession: P12175) is 511 amino acids long, which corresponds to a 61.4-kDa protein. The 55-kDa protein band we observed on Western blots of rice protein extract corresponds closely to this predicted size for MATK from GenBank. Further, competition experiments verified the specificity of our antibody to the MATK protein by successful competition of binding by the 15-amino-acid MATK antigen peptide sequence used to generate this antibody (Fig. 4B). The identity of this immunoreactive 55-kDa protein as MATK is also supported by the correlation between RNA levels for the 2.6- and 2.9-kb matK transcripts and the pattern of expression for this protein in etiolation and developmental stage experiments (Fig. 5). We propose that the additional 30- and 20-kDa protein bands immunoreactive to MATK antibody observed in rice immunoblots are the products of MATK protein proteolysis. The absence of the 30- and 20-kDa molecular mass bands in pre-immune controls and lack of these bands after addition of antigen in competition assays indicates that these bands are the MATK protein (Fig. 4). The low molecular mass of these bands, however, suggests that these are not the full-length MATK protein but most likely degraded products. Multiple bands have been observed on SDS-PAGE gels and/or immunoblots as a result of protein proteolysis even when using highly purified antibodies and protease inhibitors (Bialek et al., 1989
; Cramer et al., 1998
). We conclude, therefore, that the 55-, 30-, and 20-kDa immunoreactive bands on Western blots are the MATK protein, with the 55-kDa protein being the full-length polypeptide. Although three studies have previously reported finding a MATK protein from plant extracts (du Jardin et al., 1994
; Liere and Link, 1995
; Vogel et al., 1999
), only Vogel et al. (1999)
, using barley, identified a MATK protein close to the expected molecular mass (predicted 56 kDa, observed 60 kDa). Our findings concur with those of Vogel et al. (1999)
and demonstrate that expressed full-length MATK protein in rice is close to the predicted size.
Etiolation of matK/trnK
Protein levels for the 55-kDa MATK protein increased less than the RNA after light exposure (Fig. 5A, B). This may reflect differential regulation of expression at the RNA and protein level for matK or delayed translation of protein from RNA transcripts. Nonetheless, these results indicate that the expression of matK is influenced by light. Similarly, a five-fold increase in matK transcription was observed in a Nicotiana tabacum L. (tobacco, Solanaceae) chloroplast microarray study when tissues were grown in light (Nakamura et al., 2003
). Thus, the two studies provide independent evidence in support of light-induced matK expression and a function for MATK related to light.
Several chloroplast genes have light-induced expression (Klein and Mullet, 1990
; Klein, 1991
; Baumgartner et al., 1993
). These genes are involved in two major activities of the chloroplast: photosynthesis and chloroplast development (Mullet, 1988
; Klein and Mullet, 1990
; Klein, 1991
). Potential substrates for MATK maturase activity include RNAs from one photosynthesis-related gene, atpF (Herrmann et al., 1993
; Kostrzewa and Zetsche, 1993
; Jenkins et al., 1997
; Vogel et al., 1999
), and several other genes needed to form the chloroplast translation machinery (e.g., trnK, trnA, trnI, rpl2, and rps12 (Hess et al., 1994
; Ems et al., 1995
; Jenkins et al., 1997
; Vogel et al., 1999
). Chloroplast development requires turning on protein translation in this organelle and increases the expression of all RNAs and proteins related to the translation machinery (Baumgartner et al., 1993
). Levels of the maturase (MATK?) needed for processing introns in these transcripts should, therefore, increase with light exposure after etiolation in order to generate the needed proteins and tRNAs for photosynthesis and/or the chloroplast translation machinery.
matK RNA and protein levels during plant development
We detected matK RNA and protein at all times examined; however, the levels dropped significantly at 4 wk postgermination (Fig. 5C). This downregulation in matK expression may be associated with reduced expression of one of its proposed RNA substrates. While several genes are known to be involved directly or indirectly in the regulation of plant development (Jan et al., 2006
; Wang et al., 2006
), including the chloroplast posttranscription factor PNPase (Sauret-Güeto et al., 2006
), substrates for the proposed maturase activity of MATK mainly include tRNAs and mRNAs needed for chloroplast protein translation (Hess et al., 1994
; Ems et al., 1995
). These proposed substrates are not directly related to developmental stage. Further investigation is required to identify potential substrates related to plant development that reflect this observed downregulation for MATK at 4 wk postgermination.
matK transcription and protein expression across land plants
A matK transcript has been previously identified in the grasses O. sativa and H. vulgare (Kanno and Hirai, 1993
; Vogel et al., 1997
), tobacco (Nakamura et al., 2003
), Anthoceros formosae Steph. (Anthocerotaceae) (Kugita et al., 2003
), and A. capillus-veneris L. (Pteridaceae) (Wolf et al., 2004
). These studies, however, primarily focused on the matK/trnK gene unit as a whole and did not separate the expression of these two genes or correlate transcription data with protein expression. Our study has provided strong evidence that trnK and matK are transcribed independently of each other in rice and that the most abundant transcripts for this gene region correlate with the expression of matK, not a trnK precursor. Under this assumption, we expanded the survey for a matK transcript to 13 land plant families (14 species). A matK transcript was found in 11 plant species spanning the land plant phylogenetic tree (Table 1). These species included the orchid, S. plicata (Fig. 6A), noted previously to contain matK as a possible pseudogene (Freudenstein et al., 2004
). Interestingly, the translated amino acid sequence from the matK cDNA of this orchid did not contain any premature stop codons (GenBank accession DQ525860).
Three of the angiosperm species identified to contain matK RNA (O. sativa, S. latifolia, and A. thaliana) also contained full-length MATK protein (Fig. 6). Although these results do not demonstrate a direct relationship between matK RNA and protein expression, they strongly support that matK RNA is indicative of expressed MATK protein. While the homology of our antigen to nonmonocot angiosperms is substantially less than within the monocots (nearly 100% to closely related monocots vs. 40% to Arabidopsis), it appears that the homology between our target antigen in rice and Arabidopsis occurs at key amino acid positions sufficient for antibody binding. At present, there is no way to reliably predict the minimum amount of homology required between sequences for antibody binding. For example, only four contiguous amino acids appear to be required for cross-reactivity of the GroEL antibody from the bacteria Actinobacillus actinomycetemcomitans to human fibronectin (Yoshioka et al., 2004
). The amount of homology needed for the MATK antibody we developed in this study appears to be greater than 13% as determined by the lack of specific binding to protein from the fern P. nudum (Fig. 6B).
A full-length MATK protein was not found in extracts of the three orchids examined by Western blot. This could be due to protein proteolysis as indicated by the 23-kDa immunoreactive protein band recognized with our MATK antibody against extract of S. gracilis (Fig. 6B). On the basis of the cDNA sequence of S. plicata and detection of full-length MATK protein in several other angiosperms (Fig. 6B), this protein is likely to be translated in the orchids.
Conclusion
Establishing a functional role for MATK has important implications for plant phylogenetics, gene evolution, and plant molecular biology. The results of this study support independent transcription for the matK gene from the trnK unspliced precursor and demonstrate MATK protein expression in several plant species. Furthermore, we provide evidence of a function for MATK in plant physiology by demonstrating an influence of light and developmental stage on its expression. Maintenance of expression and functionality for matK despite its high nucleotide and amino acid substitution rate suggests that genetic buffers are in place, which constrain the evolution of this gene. Assessment of protein evolution in relation to function and gene structure is required to better understand the nature of evolutionary constraints inherent to matK and other functional genomic regions. Such insights about genes used in molecular systematics are valuable in gene choices for systematic studies and interpretations of patterns of species and gene evolution.
In addition to the importance of matK in phylogenetic and evolutionary studies, this gene also encodes a putative group II intron maturase of the chloroplast. The relationship we found between light and developmental stage and amount of matK expressed suggests important functions for this protein in plant physiology. Review of potential substrates for MATK activity supports a role for this maturase at the posttranscriptional level in essential plant activities influenced by light, such as photosynthesis. Only one study (Sauret-Güeto et al., 2006
) has previously reported a link between chloroplast posttranscription processing factors and regulation of plant developmental stage. MATK may be a second posttranscription regulatory connection between the chloroplast and plant development. To our knowledge, ours is the first study to show these functional aspects of MATK activity. Further investigations are needed to determine how MATK functions in these plant processes and to provide a more comprehensive understanding of the evolution of this unusual gene.
APPENDIX
Plant species used in this study along with their respective lineage, specimen origin, and voucher information. Vouchers for specimens are deposited in the following herbaria: DR = Technische Universität Dresden; Quandt = Herbarium Quandt (personal collection of Dietmar Quandt); and VPI = Virginia Polytechnic Institute and State University. Voucher specimens are not available for Malleola ligulata and Holcoglossum kimballianum
FOOTNOTES
1 The authors appreciate the support of this research by NSF grant EF-0431057, NSF Deep Time, Sigma Xi, Virginia Academy of Sciences, and Virginia Tech. They also thank D. Quandt for help collecting plant material and primer design, C. Neinhaus and D. Jarrell for providing plant material, and D. Wiley-Vawter and T. Wieboldt for help with voucher specimens. Special thanks to D. Bevan for assistance with peptide antigen design, S. Drysdale and S. Majumder for assistance in the laboratory, and S. Parker and the anonymous reviewers for comments on the manuscript. 
2 Author for correspondence (michelle.barthet{at}usyd.edu.au
; current address: School of Biological Sciences, University of Sydney, NSW 2006, Australia
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