| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online November 7, 2002; 10.1104/pp.008011 Plant Physiol, December 2002, Vol. 130, pp. 1837-1851 Cloning, Functional Expression, and Subcellular Localization of Multiple NADPH-Cytochrome P450 Reductases from Hybrid Poplar1Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
NADPH:cytochrome P450 reductase (CPR) provides reducing equivalents to diverse cytochrome P450 monooxygenases. We isolated cDNAs for three CPR genes (CPR1, CPR2, and CPR3) from hybrid poplar (Populus trichocarpa × Populus deltoides). Deduced CPR2 and CPR3 amino acid sequences were 91% identical, but encoded isoforms divergent from CPR1 (72% identity). CPR1 and CPR2 were co-expressed together with the P450 enzyme cinnamate-4-hydroxylase (C4H) in yeast (Saccharomyces cerevisiae). Microsomes isolated from strains expressing CPR1/C4H or CPR2/C4H enhanced C4H activities approximately 10-fold relative to the C4H-only control strain, and catalyzed NADPH-dependent cytochrome c reduction. The divergent CPR isoforms (CPR1 and CPR2/3) contained entirely different N-terminal sequences, which are conserved in other plant CPRs and are diagnostic for two distinct classes of CPRs within the angiosperms. C-terminal green fluorescent protein fusions to CPR1 and CPR2 were constructed and expressed in both yeast and Arabidopsis. The fusion proteins expressed in yeast retained the ability to support C4H activity and, thus, were catalytically active. Both CPR::green fluorescent protein fusion proteins were strictly localized to the endoplasmic reticulum in transgenic Arabidopsis. The lack of localization of either isoform to chloroplasts, where P450s are known to be present, suggests that an alternative P450 reduction system may be operative in this organelle. Transcripts for the three poplar CPR genes were present ubiquitously in all tissues examined, but CPR2 showed highest expression in young leaf tissue.
Cytochrome P450 monooxygenase
enzymes (P450s) play key roles in a wide range of oxidative metabolic
reactions in various organisms from microbes to humans. In animals,
xenobiotic detoxification and sterol biosynthesis are the major
functions of P450s. However, P450-mediated reactions in plants
encompass a much broader spectrum, including biosynthesis of
plant hormones and signal molecules, defense-related chemicals and
structural secondary natural products, and herbicide detoxification
(for review, see Werck-Reichhart et al., 2000 Most eukaryotic P450s are not self-sufficient enzymes, and their
catalytic activities strictly rely on an electron donor, NADPH:cytochrome P450 reductase (CPR; Lu et al., 1969 In animals, a single CPR is known to interact with a number of
different P450 enzymes in a given species (Simmons et al., 1985 The ER membrane is generally accepted as the primary subcellular
target for eukaryotic P450 and CPR enzyme localization, although some
P450s are present in animal mitochondria (Okuda, 1994 In vascular plants, lignin is a major natural product and its
biosynthesis requires the action of the P450 enzymes CYP73A5 (C4H;
Mizutani et al., 1997 Poplar (Populus trichocarpa × Populus
deltoides) has emerged as a model tree for molecular, genetic, and
biochemical studies (Hertzberg et al., 2001
The Poplar Genome Contains at Least Three CPR Genes Two divergent Arabidopsis CPR cDNAs (Mizutani and Ohta,
1998 The CPR1 sequence was more closely related to the Arabidopsis CPR1 gene than to the Arabidopsis CPR2 gene (Fig. 1), suggesting that other CPR isoforms more closely related to Arabidopsis CPR2 could be present in poplar. Probing the young leaf cDNA library with the Arabidopsis CPR2 cDNA at low stringency allowed us to collect a set of CPR cDNA clones that were classified into two groups by restriction enzyme digestion analysis. Sequencing a few clones from each group revealed two more full-length cDNAs (CPR216 and CPR351) that were 90% identical to each other in their coding regions and 80% identical in their 3'-untranslated regions. Two more partial clones from the CPR216 group were identical to the CPR216 in the sequenced regions. However, as was seen in the CPR1 sequence analysis, the partial nucleotide sequence alignment of five cDNA clones from the CPR351 group revealed that there were also two very similar but distinct subgroups within this group, with 97.8% nucleotide identity. The position and nature of nucleotide changes between these two subgroups were identical (data not shown), indicating that the several nucleotide differences were not caused by cloning or sequencing artifacts but derived from the poplar genome. Thus, these two very similar classes of clones are likely to represent two alleles in the CPR351 group. Assuming the presence of two distinct alleles within the CPR351 group, CPR216 is, therefore, not allelic to CPR351 in the diploid poplar genome.
The two CPR genes represented by CPR216 and CPR351 were
designated as CPR2 (accession no. AF302497) and
CPR3 (accession no. AF302498), respectively. The CPR2 and
CPR3 cDNAs possess stop codons 9 and 18 bp upstream of the first ATG
codons, respectively, suggesting that these are full-length cDNAs.
Two more putative ATG codons for CPR2 cDNA and one more ATG codon for
CPR3 cDNA are located closely downstream of the first start codon.
However, the nucleotides neighboring these ATGs deviated from the dicot start codon consensus, a(a/c) aAUGG, particularly with the occupancy of
a pyrimidine at the important Amino acid sequence alignments showed that all poplar CPR polypeptides share 70% to 77% amino acid identities to CPRs from other plants. Conserved cofactor and substrate-binding domains characteristic for the CPR, such as FMN-, FAD-, NADPH-, cytochrome c-, and P450-binding sites, were identified in all three poplar CPR polypeptides (data not shown), and their N-terminal domains contain an approximately 20-amino acid hydrophobic patch required for membrane anchoring (Fig. 1A). Sequence alignment of N-terminal ends revealed that these could be used to group angiosperm CPRs into two classes, class I and class II, based on their sequences located N terminal to the hydrophobic region. Poplar and Arabidopsis CPR1 and CPRs from spring vetch, mung bean, and opium poppy (class I) contained short N-terminal ends with several conserved amino acids. On the other hand, poplar CPR2/3, Arabidopsis CPR2, parsley CPR1 and CPRs from tobacco, pea, California poppy, and Madagascar periwinkle (class II) contained extended N-terminal ends that showed significant sequence identities. Parsley CPR2 was an exception to these groupings. The overall sequence relatedness of CPRs within each class was further confirmed by phylogenetic analysis. The same grouping pattern as depicted in Figure 1A was evident in the parsimony analysis of CPR sequences, either using full-length amino acid sequences or using N-terminally (first 59 residues) deleted sequences (Fig. 1B). Therefore, two classes of CPR isoforms with distinct, conserved N-terminal ends are present in angiosperms, which appear to have evolved at least two CPR isoforms after an ancient gene duplication. Functional Expression of CPRs in Yeast (Saccharomyces cerevisiae) To confirm the bona fide CPR activity encoded by the poplar cDNAs,
we expressed CPR1 and CPR2 in a yeast system
together with C4H. It is known that endogenous CPR activity
is a limiting factor in yeast when foreign P450s are overexpressed
(Urban et al., 1994
We used a convenient in vivo enzyme assay to evaluate C4H enzyme
activity in these strains to establish optimal cell densities for
harvesting cells and culture filtrates. Figure
3 shows that the catalytic activity of
C4H was significantly higher when co-expressed with poplar
CPR1 or CPR2 than when C4H was
expressed alone, indicative of supportive role of the CPRs for C4H
activity in yeast cells. Strains with a combination of C4H
with CPR2 showed 3 to 4 times higher conversion rate of
cinnamate to p-coumarate than those with C4H and
CPR1. The maximum level of p-coumarate was
produced at a cell density of approximately 109
cells mL
Yeast microsomal fractions were prepared from yeast cells harvested at
optimal cell densities (Fig. 3) to verify the CPR activities in vitro.
Using microsomal preparations, recombinant CPR activities were
evaluated either by measuring the formation of p-coumarate from cinnamate (C4H activity) or by measuring the reduction of cytochrome c, an artificial substrate for CPR. Patterns for the formation of p-coumarate from cinnamate in the microsomes
prepared from various yeast strains (Table
I) were very similar to those from in
vivo estimations of C4H activity. In addition, when reduction of
cytochrome c was measured in vitro, the microsomal fractions from
CPR1- and CPR2-expressing yeast showed
approximately 6- and 3-fold higher cytochrome c reduction activities,
respectively, than yeast transformed with C4H alone. The
reduction of cytochrome c by recombinant CPR1 and CPR2 protein was
dependent on NADPH, but not on NADH, as is known for other CPRs
(Koopmann and Hahlbrock, 1997
Characterization of C4H/CPR Dual Expression in Yeast It was evident from the in vivo and in vitro assays that both CPR1 and CPR2 enhance C4H activity. However, in both cases, C4H activity was consistently higher when associated with CPR2 than with CPR1. These results led us to further investigate whether the higher C4H activity is related to a relatively higher affinity of C4H toward the CPR2 isoform or simply a higher level of C4H protein in the C4H/CPR2 strains. Total P450 content in microsomes can be quantified by CO differential
absorption spectroscopy (Omura and Sato, 1964 Subsequently, the expression level of C4H in differently transformed yeast strains was compared by semiquantitative immunoblot analysis and by northern-blot analysis. For immunoblot analysis, the same microsomal proteins, independently prepared and previously used for the enzyme assays, were fractionated by SDS-PAGE, and FLAG and c-Myc monoclonal antibodies used to detect specific recombinant proteins of predicted molecular weights on blots (Fig. 4A). Unexpectedly, C4H protein levels varied significantly in the different strains, although the same Gal-10 promoter in an identical vector background drove the expression of C4H in all cases. C4H levels were consistently lower in strains co-expressing CPRs than strains expressing C4H alone, and were lower in CPR1-co-expressing strains than in CPR2 co-expressing strains. C4H transcript levels in the different yeast strains showed a similar pattern, with highest amount of C4H expression in strains expressing C4H alone, and lower amounts in strains co-expressing CPR1 and CPR2 (Fig. 4B).
Based on quantitation of immunodetectable protein levels, the relative C4H activity in yeast expressing C4H alone was approximately 10% or less than that in the C4H/CPR dual expressers (Table I), and within the dual expressers, the amount of C4H transcript and protein was generally proportional to the C4H activities previously measured by in vivo and in vitro enzyme assays. Thus, considering differences in C4H protein amount in different strains, the difference in the relative C4H activities between yeast strains expressing C4H/CPR1 and C4H/CPR2 was small (less than 2-fold; Table I). The enhanced C4H activity in the yeast strain co-expressing CPR2 was largely the result of elevated C4H transcript and protein levels in this strain relative to the CPR1-expressing strain, and not the result of enhanced ability of CPR2 to support C4H activity in yeast. Two Distinct CPR Isoforms Are Localized to the ER The three poplar CPR isoforms contain highly conserved amino acid
sequences in the FMN-, FAD-, cytochrome c-, and NADPH-binding sites
(data not shown). However, the CPR1 and CPR2/3 cDNAs encode two
entirely different N termini, each of which has higher identity to its
apparent isoform ortholog in Arabidopsis than to the other poplar
isoform (Fig. 1). This could suggest important roles of the
evolutionarily conserved N-terminal sequences in directing CPR1 and
CPR2/3 to distinct subcellular locations, as similarly suggested for
Arabidopsis CPRs by Urban et al. (1997)
To definitely determine the subcellular localization of the poplar
CPRs, we fused GFP to the C termini of the predicted full-length CPR1
polypeptide and to two versions of CPR2, and expressed them in
Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. As shown in Figure 5A, CPR2 and CPR3 contain very similar
N termini; therefore, CPR3 was not used in these studies. CPR21 is a
full-length version of CPR2, whereas CPR22 was truncated such that its
second ATG is the translation start codon, resulting in deletion of
five Ser (Fig. 5A). To meet the nucleotide preference for the
translation initiation (Joshi et al., 1997 A total of >80 independently transformed T1 and T2 Arabidopsis seedlings for each construct were prescreened for the presence of green fluorescence by fluorescence microscopy. Most of the kanamycin-resistant plants presented weak GFP signals associated with the perinuclear membrane in the guard cells but showed no fluorescence in the epidermal cells (data not shown). Several Arabidopsis lines containing each construct showed detectable green fluorescence in the epidermis and some cortex cells. Immunoblot analysis of the microsomal proteins from these Arabidopsis seedlings demonstrated that intact fusion proteins were expressed in these Arabidopsis lines (Fig. 5B). Confocal microscopy showed that all three different GFP-fused CPRs were
strictly localized to the ER membrane in the epidermal cells of
transgenic plants (Fig. 5, C-E). The GFP localization patterns
observed here were identical to those observed for poplar C4H::GFP (Ro et al., 2001 Functional verification of GFP fusion protein activity can be used to exclude potential localization artifacts caused by nonfunctional GFP fusions. For this purpose, the CPR::GFP constructs were recloned into a pESC yeast expression vector already containing the C4H, and co-expressed with C4H in yeast. In vivo analysis of C4H activities in the absence or presence of the CPR::GFP-fusion proteins in these strains was used to assay CPR::GFP function. Figure 6A shows that yeast transformed with the CPR::GFP constructs supported the C4H reaction at significantly higher rates relative to C4H activity in control cells expressing only C4H. To validate this observation, the presence and relative amounts of the CPR::GFP and C4H proteins were estimated by immunoblot analysis using anti-GFP or anti-FLAG antibodies to detect CPR::GFPs and C4H in yeast microsomal proteins (Fig. 6B). As previously observed, the highest amount of C4H was detected in the yeast strain expressing C4H alone, although this strain had the lowest C4H activity. These data demonstrate that CPR::GFP enzymes are active and that the C-terminal GFP fusions do not significantly disrupt interaction with C4H in vivo.
CPR Expression Patterns in Poplar The mRNA accumulation patterns of poplar CPRs in various tissues and in poplar cultured cells with or without elicitor treatment were examined by semiquantitative reverse transcriptase (RT)-PCR analysis. This RT-PCR analysis employed CPR gene-specific primers and 18S-specific primers as internal controls for RNA amount. PCR cycle number was optimized for each gene to ensure that amplification endpoints were in the logarithmic phase. CPR1-specific primers were designed in the coding region of CPR1. For CPR2 and CPR3, a common forward primer in the 5'-coding region of CPR2/3 and distinct reverse primers each for CPR2 or CPR3 in the 3'-untranslated regions were designed. Figure 7A shows that these gene-specific primer pairs were able to distinguish different CPR cDNAs used as templates, and, thus, are gene specific. However, using poplar genomic DNA as templates, the CPR1-specific primers did not amplify any PCR product, whereas the CPR2 and CPR3 primers showed about 800 bp longer PCR products than those calculated from the corresponding CPR cDNAs. These data suggest that the CPR1 primers span an exon-intron junction or flank a very long intron in genomic DNA (resulting in no PCR products). The longer genomic fragments amplified by CPPR2/3 primers relative to the cDNAs likely arose because of the presence of introns in poplar CPRs as shown in Arabidopsis CPR (17 introns for each Arabidopsis isoform). Therefore, the primer sets for each CPR isoform were not only gene specific, but also distinguished CPR cDNA amplicons from those of genomic DNA.
We have documented previously the expression pattern of the
poplar PAL2 gene (PAL7 cDNA) by northern-blot analysis
(Subramaniam et al., 1993 Amplified 18S ribosomal RNA fragments interfered with RT-PCR using CPR2
and CPR3 primers, necessitating amplification of transcripts from these
two genes without this internal control. Figure 7B shows that the
highest amount of CPR2 transcript was detected in young
leaf, and that its expression in cultured poplar cells was barely
detectable. The CPR3 transcripts were also ubiquitously present in
tissues and cultured cells as observed in CPR1. The CPR3 gene appeared to have the strongest expression level
among the CPR genes because, with the same amount of total
RNA, it required about five fewer PCR cycles than CPR1 and
CPR2. We have shown previously a strong induction of genes
involved in phenylpropanoid metabolism, such as PAL,
C4H, and 4CL, from the poplar cell cultures treated with an elicitor derived from cell walls of Fusarium
oxysporum (Moniz de Sa et al., 1992
Multiple Isoforms of CPR in Poplar As is the case for other poplar phenylpropanoid genes such as
PAL, 4CL, and C4H (Subramaniam
et al., 1993 The identification of two divergent classes of poplar CPR
genes (CPR1 and CPR2/3) shows that this plant
contains two distantly related CPR genes very similar to
those in Arabidopsis (Urban et al., 1997 The occurrence of multiple CPRs in plants may reflect the
diversity of P450s that catalyze reactions for the synthesis of a wide
range of primary and secondary metabolites essential for plant growth
and development (Werck-Reichhart et al., 2000 The authentic activities of recombinant poplar CPR isoforms were verified by expression in yeast. The dual expression vector employed allowed convenient co-expression of C4H and CPR in a single yeast strain. The Gal-1 and Gal-10 promoters controlling C4H and CPR expression are strictly regulated by Glc/Gal at the level of transcription and the promoters comparably induce reporter genes (information supplied by Stratagene). Thus, we expected that similar levels of both recombinant proteins would accumulate. This was expected to simplify in vivo evaluation of differential ability of CPR to support C4H activity without membrane reconstitution in vitro using individually prepared recombinant proteins. However, we unexpectedly found unequal amounts of recombinant C4H and CPR in different strains (Fig. 4). The significantly decreased C4H recombinant protein levels relative to
CPR in C4H/CPR dual-expressing strains could result from the presence
of C4H codons unfavorable for use in yeast. Codon use in both
CPR1 and CPR2 mRNAs does not severely deviate from that in yeast, but the predicted C4H mRNA contains a few unfavorable codons for yeast expression such as CTC (Leu) and AGG
(Arg). We recovered >100 pmol C4H mg Although different levels of recombinant proteins that accumulated
complicated analysis, the data here in general show that both CPR
isoforms could nearly equally support the activity of C4H, the best
characterized A-type P450 enzyme. This is in agreement with findings
reported from other plants (Urban et al., 1997 Two Divergent CPR Isoforms Are Localized to the ER Sequence comparison of the CPR isoforms from poplar and
Arabidopsis revealed striking cross species identities in the N termini within the same CPR group. Other angiosperm species also contain CPRs
that group either with class I or II CPR isoform from poplar and
Arabidopsis not only in their N-terminal sequences but by phylogenetic
analysis of complete coding amino acid sequences (Fig. 1). The overall
length of N termini and even many amino acids were conserved in each
class of CPR in an isoform-specific manner. This conservation of this
primary sequence suggests functional relevance of the N-terminal domain
such that it has been maintained through evolution in distantly related
species. One plausible explanation for this is that the different N
termini contain information for the different subcellular destination
of each CPR isoform. In particular, poplar CPR2/3 and its
ortholog Arabidopsis CPR2 possess an extended N terminus
with high Ser/Thr content, generally believed to be required for
chloroplast targeting (von Heijne et al., 1989 As was shown in C4H::GFP localization studies (Ro et
al., 2001 The CPR::GFP localization results were further substantiated by the ability of CPR::GFP to support C4H activity in yeast, indicating that the GFP domain of the fusion enzyme does not significantly interfere with CPR-C4H interaction in living cells, and that correct protein folding and orientation of CPR on the ER is maintained in the fusions. In contrast, the CPR::GFP fusion enzymes were incapable of supporting reduction of cytochrome c in microsomes in an in vitro assay (data not shown). The observed preferential reduction of C4H by CPR::GFP compared with cytochrome c may be because of the fact that cytochrome c interacts with CPR from the aqueous phase in the in vitro assay, whereas C4H interacts with CPR in a tight membrane-bound form through the ER. The membrane anchoring domains of P450s and CPR may stabilize the P450-CPR association either by direct N-terminal hydrophobic interactions or by conferring the favorable structural conformations for P450-CPR interactions. Alternatively, the GFP fusion may result in steric hindrance that prevents CPR interaction with cytochrome c in the presence of C4H. CPR-Independent Electron Transport Path to P450s in Chloroplasts? Arabidopsis P450 enzymes have been identified that are likely to
be chloroplast localized based on the high Ser/Thr content of their N
termini (Watson et al., 2001 In the apparent absence of a chloroplast-targeted CPR species, it is
likely that a non-homologous electron donor system is present in
chloroplast to support P450 activity in this organelle. In animals, a
specific mitochondrial electron donor system composed of two proteins,
adrenodoxin and adrenodoxin reductase, supplies electrons from NADPH to
mitochondrial P450s (Omura et al., 1966 CPR Expression Patterns in Poplar RT-PCR analysis detected all three CPR transcripts in
several poplar tissues, but young leaves always showed higher levels of
CPR transcript accumulation. CPR2 showed similar
but slightly different expression patterns from CPR1 and
CPR3. Accumulation of CPR2 transcripts was more
predominant in young leaf, and CPR2 expression was low in
both elicitor-treated and non-treated poplar cell cultures. Although
speculative, this suggests a specific role for CPR2 in
poplar young leaves in which other phenylpropanoid genes are also
highly expressed (Subramaniam et al., 1993 There was no significant induction of any of the three CPR
genes in an elicitor-treated cell culture harvested 2 h after
elicitor treatment, when PAL and 4CL mRNAs are
maximally expressed (Moniz de Sa et al., 1992
Isolation of CPR cDNA Clones A cDNA library from hybrid poplar (Populus
trichocarpa × Populus deltoides) H11-11
young leaf or xylem mRNAs was previously constructed in To obtain a full-length CPR cDNA, forward (5'-GCTAGTGACCACGTTTCAAGACAAC-3') and reverse (5'-AGCTTTAATTCTACCTTGACACCTGG-3') primers specific to the 3'- and 5'-untranslated regions of CPR122 and CPR181 partial cDNA clones were used to PCR amplify a full-length CPR cDNA from a cDNA pool of H11-11 xylem in a 50-µL reaction mixture containing 20 pmol each primer, 2.5 units of Pyrococcus furiosus polymerase (Stratagene), 100 ng of xylem cDNA template, 0.2 mM dNTP, and 2 mM Mg2+. The PCR conditions used were: one cycle of 3 min at 94°C, 25 cycles of 1 min at 94°C, 1 min at 62°C, and 3 min at 72°C. The PCR product was incubated with 5 units of Taq polymerase (Life Technologies/Gibco-BRL) at 72°C for 10 min to add overhang adenine nucleotides. The 2.5-kb PCR products were cloned into a pCR2.1 vector using a PCR TA cloning kit (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. This CPR clone was named CPR1. Other CPR isoforms were recovered from the poplar young leaf cDNA library, using an AR2 coding region as a hybridization probe under low-stringency conditions, as described above. As a result, 38 positive clones were identified, which were classified into two groups based on restriction fragment analyses using HindIII and PvuII. The longest clones from each group were completely sequenced, and named CPR2 and CPR3. Construction of C4H, CPR, and CPR::GFP in the pESC-Leu Vector Coding regions for C4H, CPR1, and CPR2 were amplified by PCR using the Pwo DNA polymerase (Roche Applied Science, Indianapolis). Gene-specific primers with NotI, SalI, or ApaI restriction enzyme sites (underlined) used for PCR were: for the C4H coding region, a forward primer, 5'-ATAAGACTGCGGCCGCATCATGGATCTCCTTCTCCTG-3' and a reverse primer, 5'-AAGTAGTAGCGGCCGCAAAGGACCTTGGCTTTGCAACAATAG-3'; for the CPR1 coding region, a forward primer, 5'-TACAGCGGGCCCAGGATGAGTTCAGGTGGTTCAAAT-3' and a reverse primer, 5'-TACAGCGTCGACCCAGACATCTCTTAGATACCGTCC-3'; and for the CPR2, a forward primer, 5'-TACAGCGGGCCCAACATGCAATCATCAAGCAGCTCG-3' and a reverse primer, 5'-TACAGCGTCGACCCATACACGCAGATACCTGC-3'. The pESC-Leu yeast dual expression vector (Stratagene) was used to clone and express these genes. The PCR fragment for C4H was digested with NotI and inserted into the corresponding site behind the Gal-10 promoter in-frame with the FLAG epitope in the correct orientation. The PCR fragments for CPR1 or CPR2 were digested by SalI and ApaI and inserted into the corresponding sites behind the Gal-1 promoter in-frame with the c-Myc epitope. Thus, three different forms of the pESC-Leu expression vector were prepared to contain C4H alone, C4H and CPR1, or C4H and CPR2. A series of CPR::GFP genes in the pBin19 vector (see below) were recloned into a pESC dual expression vector along with C4H. The CPR1::GFP fusion gene constructed in the pBin19 vector was used as a template in a PCR amplification. The coding sequence for CPR1::GFP was amplified using Pwo polymerase, a forward primer 5'-TACAGCGGGCCCAGGATGAGTTCAGGTGGTTCAAAT-3', and a reverse primer, 5'-TACAGCGTCGACTTATTTGTATAGTTCATCCATGCC-3'. The PCR products were digested by ApaI and SalI, and inserted into the ApaI and XhoI sites of the pESC-Leu vector in which the C4H had previously been cloned. For yeast expression of the GFP-fusion of CPR21 and CPR22, DNA fragments containing a CPR21::GFP or CPR22::GFP coding sequence were isolated by ApaI/XhoI digestion from the pSL1180-based CPR::GFP constructs. These fragments were directionally cloned into the ApaI/XhoI sites of a pESC-Leu vector in which the C4H had been cloned previously. Yeast Culture Media, Transformation, and Gene Induction Untransformed YPH499 strain (MATa, ura3-52, lys2-801, ade2-101,
trp1- For C4H and CPR gene induction in the
YPH499 strain, the transformed yeast strains were subcultured in 3 mL
of SD-Leu dropout medium overnight and 250 mL of SD-Leu dropout medium
was subsequently inoculated by a 1:100 (v/v) dilution. Yeast
cells were cultured at 28°C on a shaker at 150 rpm for 12 to
15 h until cultures reached mid-log phase (approximately 3-5 × 107 cells mL Enzyme Assays C4H enzyme assays were performed as described previously
(Ro et al., 2001 For in vivo enzyme assays in yeast, the number of yeast cells,
estimated by the spectrophotometric optical density at 600 nm
(OD600), was used to normalize the enzyme activity data
obtained from in vivo enzyme assays. An OD600 of 1 was
determined to be equivalent to 3.0 × 107 cells
mL Microsomal Protein Preparation Microsome preparation from yeast was performed as described
(Pompon et al., 1996 Construction of CPR::GFP Fusions in the pBin19 Vector A C4H::GFP fusion construct had been
generated previously in the pSL1180 vector (Ro et al.,
2001 Plant Growth Conditions and Transformation Arabidopsis ecotype Columbia was germinated and grown on medium
containing a 0.5× Murashige and Skoog basal salt mixture (Sigma) for 1 week at 20°C under constant light, and the seedlings were then
transferred to soil (Redi-Earth, W.R. Grace and Co., Ontario, Canada)
under the same growth conditions. Plants were grown until abundant
immature floral clusters had formed, and then transformed by the floral
dip method (Clough and Bent, 1998 Fluorescence and Confocal Microscopy Analysis To detect fluorescent signals in CPR::GFP-transformed Arabidopsis lines, at least 80 independently transformed seedlings (3-5 d old) were prescreened by fluorescence microscopy using a light microscope equipped with a GFP filter set, and the strongest fluorescent lines were further analyzed by a Radiance confocal microscope (Bio-Rad Laboratories, Hercules, CA). Seedling samples were gently squeezed between cover and slide glass with a drop of distilled water, and a line of a 488-nm argon laser (10%-20%) was used as an excitation light source. Green and red fluorescent signals from GFP and chloroplasts were separately collected through the HQ515/30 or E600LP barrier filter, respectively. NIH Image software (National Institutes of Health, Bethesda, MD) was used to process the confocal microscopy data. Five to 10 sections at 0.2-µm intervals were overlapped to enhance the fluorescence signals. Collected images were further processed by the NIH Image or PhotoShop 5.0 software. SDS-PAGE and Immunoblots Protein concentrations were quantified by the Bradford method using BSA as a standard. Microsomal protein samples (2 µg for yeast and 20 µg for Arabidopsis) were separated on 7.5% (w/v; for GFP-fused proteins) or 10% (w/v; for non-GFP-fused proteins) polyacrylamide gels, and either stained with Coomassie Blue or transferred to PVDF membrane (Amersham-Pharmacia Biotech, Uppsala) for immunoblot analysis. Primary antibodies used, depending on the target proteins, were anti-FLAG (Stratagene), anti-cMyc (a gift from Dr. Michael Gold, Department of Microbiology and Immunology, University of British Columbia), or anti-GFP (Sigma) monoclonal antibodies. Immunodetection was performed using the ECL system (Amersham-Pharmacia Biotech). For simultaneous detection of C4H and CPR, sequential immunodetection was employed. Duplicate blots were prepared and reacted with FLAG or cMyc primary antibodies independently for 30 min, followed by secondary antibody binding and ECL-based signal detection in standard conditions. Each primary antibody identified a protein band of the predicted size, and no cross-reaction to other proteins was detected. The two blots were then washed with 1× Tris-buffered saline buffer supplemented with 0.05% (v/v) Tween 20 for 1 h, and reacted again with the reciprocal second primary antibody for 1 h. After incubation with secondary antibody at a 1:5,000 (v/v) dilution for 1 h, signals were detected as before. X-ray films were exposed to a level of under-saturation and the intensities of signals on the film were quantified by a densitometer (Bio-Rad Laboratories). The dual (FLAG and cMyc) and single signals (FLAG or cMyc) detected in the duplicated blots during this experiment showed very similar patterns. Nucleic Acid Isolation and Northern Blots For isolation of poplar genomic DNA, 1 g of frozen poplar
(clone H11-11) young leaf was ground to a fine powder in liquid nitrogen, and the Nucleon PhytoPure beads (Amersham-Pharmacia Biotech)
were used to retrieve DNA, according to the manufacturer's instructions. For RNA isolation from poplar cell culture and organs, the Nucleon PhytoPure method (Kiefer et al., 2000 RT-PCR Expression Analysis Gene-specific primers used to amplify a cDNA fragment of PAL1/2 were: a forward primer, 5'-GTTGCATCCATTGCTGGTCATGATAC-3' and a reverse primer, 5'-GAATCCAGATTCAATGCCAGCTGCTT-3'; for CPR1, a forward primer, 5'-CCTAGCGAGGCAGATAGACTCAAGT-3' and a reverse primer, 5'-TAGTTCATATCTCTGCTGCTCTATC-3'; for CPR 2, a forward primer, 5'-TCATTATGATTGGCCCTGGAACTGGT-3' and a reverse primer, 5'-CAAGGCTTCAACGGAGTTAACTTTTG-3'; and for CPR3, the same forward primer as for the CPR2 and a reverse primer, 5'-GGCTTCGGTATTTATAGAGTAAACTTT-3'. The specificities of the CPR primers were verified by PCR using 1 ng of PAL and CPR cDNA or 100 ng of poplar genomic DNA as templates. Taq polymerase (Life Technologies/Gibco-BRL) was used with 30 cycles of PCR under the following conditions: 30 s of denaturation at 94°C, 30 s of annealing at 62°C, and 1 min of polymerization at 72°C. OneStep RT-PCR reagent in a 25-µL final volume was used for optimization and experiments according to the manufacturer's protocol (Qiagen USA, Valencia, CA). Using young leaf total RNA, PCR cycle numbers were optimized for each gene such that product accumulation was in the exponential range. In the optimized cycle-range (20-25 cycles), a 7:3 ratio of 18S ribosomal RNA primers and competimers (QuantumRNA universal 18S internal standard, Ambion) was determined to be optimal, and 1 µL of this mixture was added to the final 25-µL reaction volume. Using gene-specific primers and 100 ng of total RNA from various tissues |
TOP
ABSTRACT
INTRODUCTION
3 position (with A in ATG as +1;
Joshi et al., 1997
1.
ZAPII
(Subramaniam et al., 1993
63, his3-
-mercaptoethanol. The suspension was centrifuged at
13,000g for 5 min at 4°C and the supernatant was
recentrifuged at 100,000g for 30 min at 4°C in a
bench-top ultracentrifuge (Beckman Instruments, Fullerton, CA). The pellets were redissolved in 50 µL of Tris buffer (pH 7.5) containing 20% (w/v) glycerol.