Plastid Transit Peptide Research Articles

Molecular cloning, characterization and expression analysis of the gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Aquilaria sinensis (Lour.) Gilg.

The major constituents of agarwood oils are sesquiterpenes that are obtained from isoprenoid precursors through the plastidial methylerythritol phosphate (MEP) pathway and the cytosolic mevalonate pathway. In this study, a novel full-length cDNA of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), which was the second key enzyme in the plastid MEP pathway of sesquiterpenes biosynthesis was isolated from the stem of Aquilaria sinensis (Lour.) Gilg by the methods of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique for the first time, and named as AsDXR. The full-length cDNA of AsDXR was 1768 bp, containing a 1437 bp open reading frame (ORF) encoding a polypeptide of 478 amino acids with a molecular weight of 51.859 kD and the theoretical isoelectric point of 6.29. Comparative and bioinformatic analysis of the deduced AsDXR protein showed extensive homology with DXRs from other plant species, especially Theobroma cacao and Gossypium barbadense, and contained a conserved transit peptide for plastids, and extended pro-rich region and a highly conserved NADPH-binding motif owned by all plant DXRs. Southern blot analysis indicated that AsDXR belonged to a small gene family. Tissue expression pattern analysis revealed that AsDXR expressed strongly in root and stem, but weakly in leaf. Additionally, AsDXR expression was found to be activated by exogenous elicitor of MeJA (methyl jasmonate). The contents of three sesquiterpenes (α-guaiene, α-humulene and Δ-guaiene) were significantly induced by MeJA. This study enables us to further elucidate the role of AsDXR in the biosynthesis of agarwood sesquiterpenes in A. sinensis at the molecular level.

Molecular cloning and characterization of a heme oxygenase1 gene from sunflower and its expression profiles in salinity acclimation

Heme oxygenase1 (HO1) is involved in protecting plants from environmental stimuli. In this study, a sunflower (Helianthus annuus L.) HO1 gene (HaHO1) was cloned and sequenced. It was confirmed that HaHO1 encodes a precursor protein of 32.93 kDa with an N-terminal plastid transit peptide which was validated by subcellular localization. The amino acid sequence of HaHO1 shared high homology with other plant HO1s. The predicted three-dimensional structure showed a high degree of structural conservation as compared to the known HO1 crystal structures. Phylogenetic analysis revealed that HaHO1 clearly grouped with the plant HO1-like sequences. Moreover, the purified recombinant mature HaHO1 expressed in Escherichia coli exhibits HO activity. Thus, it was concluded that HaHO1 encodes a functional HO1 in sunflower. Additionally, HaHO1 gene was ubiquitously expressed in all tested tissues, and induced differentially during different growth stages after germination, and could be differentially induced by several stresses and hemin treatment. For example, a pretreatment with a low concentration of NaCl (25 mM) could lead to the induction of HaHO1 gene expression and thereafter a salinity acclamatory response. Above cytoprotective effect could be impaired by the potent HO1 inhibitor zinc protoporphyrin IX (ZnPPIX), which was further rescued by the addition of 50% carbon monoxide aqueous solution (in particular) or bilirubin, two catalytic by-products of HO1, respectively. Similarly, a HO1 inducer, hemin, could mimic the salinity acclamatory response. Together, these findings strongly suggested that the up-regulation of HaHO1 might be required for the observed salinity acclimation in sunflower plants.

Overexpression, purification and enzymatic characterization of a recombinant plastidial glucose-6-phosphate dehydrogenase from barley (Hordeum vulgare cv. Nure) roots

In plant cells, the plastidial glucose 6-phosphate dehydrogenase (P2-G6PDH, EC 1.1.1.49) represents one of the most important sources of NADPH. However, previous studies revealed that both native and recombinant purified P2-G6PDHs show a great instability and a rapid loss of catalytic activity. Therefore it has been difficult to describe accurately the catalytic and physico-chemical properties of these isoforms.The plastidial G6PDH encoding sequence from barley roots (Hordeum vulgare cv. Nure), devoid of a long plastidial transit peptide, was expressed as recombinant protein in Escherichia coli, either untagged or with an N-terminal his-tag. After purification from both the soluble fraction and inclusion bodies, we have explored its kinetic parameters, as well as its sensitivity to reduction.The obtained results are consistent with values determined for other P2-G6PDHs previously purified from barley roots and from other land plants. Overall, these data shed light on the catalytic mechanism of plant P2-G6PDH, summarized in a proposed model in which the sequential mechanism is very similar to the mammalian cytosolic G6PDH.This study provides a rational basis to consider the recombinant barley root P2-G6PDH as a good model for further kinetic and structural studies.

Tandem Repeats, High Copy Number and Remarkable Diel Expression Rhythm of Form II RuBisCO in Prorocentrum donghaiense (Dinophyceae)

Gene structure and expression regulation of form II RuBisCO (rbcII) in dinoflagellates are still poorly understood. Here we isolated this gene (Pdrbc) and investigated its diel expression pattern in a harmful algal bloom forming dinoflagellate Prorocentrum donghaiense. We obtained cDNA sequences with triple tandem repeats of the coding unit (CU); the 5′ region has the sequence of a typical dinoflagellate plastid gene, encoding an N-terminus with two transmembrane regions separated by a plastid transit peptide. The CUs (1,455 bp except 1464 bp in last CU) are connected through a 63 bp spacer. Phylogenetic analysis showed that rbcII CUs within species formed monophyletic clusters, indicative of intraspecific gene duplication or purifying evolution. Using quantitative PCR (qPCR) we estimated 117±40 CUs of Pdrbc in the P. donghaiense genome. Although it is commonly believed that most dinoflagellate genes lack transcriptional regulation, our RT-qPCR analysis on synchronized cultures revealed remarkable diel rhythm of Pdrbc expression, showing significant correlations of transcript abundance with the timing of the dark-to-light transition and cell cycle G2M-phase. When the cultures were shifted to continuous light, Pdrbc expression remained significantly correlated with the G2M-phase. Under continuous darkness the cell cycle was arrested at the G1 phase, and the rhythm of Pdrbc transcription disappeared. Our results suggest that dinoflagellate rbcII 1) undergoes duplication or sequence purification within species, 2) is organized in tandem arrays in most species probably to facilitate efficient translation and import of the encoded enzyme, and 3) is regulated transcriptionally in a cell cycle-dependent fashion at least in some dinoflagellates.

Identification and characterization of a novel chloroplast/mitochondria co-localized glutathione reductase 3 involved in salt stress response in rice

Glutathione reductases (GRs) are important components of the antioxidant machinery that plants use to respond against abiotic stresses. In rice, one cytosolic and two chloroplastic GR isoforms have been identified. In this work, we describe the cloning and characterization of the full-length cDNA encoding OsGR3, a chloroplast-localized GR that up to now was considered as a non-functional enzyme because of assumed lack of N-terminal conserved domains. The expression of OsGR3 in E. coli validated that it can be translated as a protein with GR activity. OsGR3 shows 76 and 53% identity with OsGR1 (chloroplastic) and OsGR2 (cytosolic), respectively. Phylogenetic analysis revealed 2 chloroplastic GRs in Poaceae species, including rice, sorghum and brachypodium, but only one chloroplastic GR in dicots. A plastid transit peptide is located at the N terminus of OsGR3, and genetic transformation of rice with a GR3-GFP fusion construct further confirmed its localization in chloroplasts. Furthermore, OsGR1 and OsGR3 are also targeted to mitochondria, which suggest a combined antioxidant mechanism in both chloroplasts and mitochondria. However, both isoforms showed a distinct response to salinity: the expression of OsGR3 but not OsGR1 was induced by salt stress. In addition, the transcript level of OsGR3 was greatly increased with salicylic acid treatment but was not significantly affected by methyl jasmonate, dehydration or heat shockstress. Our results provide new clues about the possible roles of functional OsGR3 in salt stress and biotic stress tolerance.

Identification and differential expression of two isogenes encoding 1-deoxy-d-xylulose 5-phosphate reductoisomerase in Glycine max

Plant 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) were considered to be encoded by single copy. In this study, we successfully isolated two DXR isogenes, designated GmDXR1 and GmDXR2, from soybean. The multicopy nature of DXRs in soybean was supported by Southern blot. Alignment of the two GmDXRs showed the presence of the N-terminal transit peptide for plastids, the conserved cleavage site, proline-rich region, and the NADPH-binding motif. Phylogenetic analysis revealed GmDXR1 and GmDXR2 belonged to different branches of angiosperm DXRs clusters. Expression pattern analysis indicated that GmDXR1 was expressed in all analyzed organs except the pod walls, with the highest level in seeds, whereas no message was detected for GmDXR2. In response to heat stress, GmDXR1 showed declined transcript levels in the experiments; in contrast, GmDXR2 was highly responsive, with mRNA accumulation peaking at 6 h after treatment. We also demonstrated that both GmDXRs were localized in chloroplasts. Overexpression of GmDXRs induced increased contents of various isoprenoids (chlorophyll, carotenoids, and gibberellins), but with reduced level of ABA, indicating that GmDXRs participate in the control of differential isoprenoid biosynthesis of the MEP pathway. This work provides a new insight into the wide presence of multicopies of DXR enzyme in plants.

The Arabidopsis Ortholog of Rice DWARF27 Acts Upstream of MAX1 in the Control of Plant Development by Strigolactones

Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. In Arabidopsis (Arabidopsis thaliana), SL biosynthesis requires the sequential action of two carotenoid cleavage dioxygenases, MORE AXILLARY GROWTH3 (MAX3) and MAX4, followed by a cytochrome P450, MAX1. In rice (Oryza sativa), the plastid-localized protein DWARF27 (OsD27) is also necessary for SL biosynthesis, but the equivalent gene in Arabidopsis has not been identified. Here, we use phylogenetic analysis of D27-like sequences from photosynthetic organisms to identify AtD27, the likely Arabidopsis ortholog of OsD27. Using reverse genetics, we show that AtD27 is required for the inhibition of secondary bud outgrowth and that exogenous application of the synthetic SL GR24 can rescue the increased branching phenotype of an Atd27 mutant. Furthermore, we use grafting to demonstrate that AtD27 operates on a nonmobile precursor upstream of MAX1 in the SL biosynthesis pathway. Consistent with the plastid localization of OsD27, we also show that AtD27 possesses a functional plastid transit peptide. We demonstrate that AtD27 transcripts are subject to both local feedback and auxin-dependent signals, albeit to a lesser extent than MAX3 and MAX4, suggesting that early steps in SL biosynthesis are coregulated at the transcriptional level. By identifying an additional component of the canonical SL biosynthesis pathway in Arabidopsis, we provide a new tool to investigate the regulation of shoot branching and other SL-dependent developmental processes.

Isolation of mitochondrial and plastid ftsZ genes and analysis of the organelle targeting sequence in the diatom Chaetoceros neogracile (Diatoms, Bacillariophyceae)

SUMMARYMitochondria and plastids multiply by division in eukaryotic cells. Recently, the eukaryotic homolog of the bacterial cell division protein FtsZ was identified and shown to play an important role in the organelle division process inside the inner membrane. To explore the evolution of FtsZ proteins, and to accumulate data on the protein import system in mitochondria and plastids of the red algal lineage, one mitochondrial and three plastid ftsZ genes were isolated from the diatom Chaetoceros neogracile, whose plastids were acquired by secondary endosymbiotic uptake of a red alga. Protein import into organelles depends on the N‐terminal organelle targeting sequences. N‐terminal bipartite presequences consisting of an endoplasmic reticulum signal peptide and a plastid transit peptide are required for protein import into diatom plastids. To characterize the organelle targeting peptides of C. neogracile, we observed the localization of each green fluorescent protein‐tagged predicted organelle targeting peptide in cultured tobacco cells and diatom cells. Our data suggested that each targeting sequences functioned both in tobacco cultured cells and diatom cells.

Characterization of a Heme Oxygenase–1 from Tobacco That Is Involved in Some Abiotic Stress Responses

Heme oxygenase–1 (HO1) is a stress protein induced by a variety of oxidative challenges. In this study, an NtHO1 gene coding for a tobacco (Nicotiana tabacum L.) HO1 was identified. The NtHO1 gene encodes an NtHO1 precursor of 32.1 kDa with a putative N-terminal plastid transit peptide. The three-dimensional structure of NtHO1 was modeled, and it showed a high degree of structural conservation compared with the known animal and bacterial HO1 crystal structures. Phylogenetic analysis revealed that NtHO1 clearly groups with the HO1-like sequences. The recombinant mature NtHO1 protein expressed heterologously in Escherichia coli was active in the conversion of heme to biliverdin IXα. The result of subcellular localization of NtHO1 confirmed the presence of a functional transit peptide and implied that the NtHO1 gene product is at least localized in the chloroplast. Expression analysis showed that NtHO1 was expressed in all tissues tested. Importantly, we found that NtHO1 expression could be induced by osmoti...

Plastid Located WHIRLY1 Enhances the Responsiveness of Arabidopsis Seedlings Toward Abscisic Acid

WHIRLY1 is a protein that can be translocated from the plastids to the nucleus, making it an ideal candidate for communicating information between these two compartments. Mutants of Arabidopsis thaliana lacking WHIRLY1 (why1) were shown to have a reduced sensitivity toward salicylic acid (SA) and abscisic acid (ABA) during germination. Germination assays in the presence of abamine, an inhibitor of ABA biosynthesis, revealed that the effect of SA on germination was in fact caused by a concomitant stimulation of ABA biosynthesis. In order to distinguish whether the plastid or the nuclear isoform of WHIRLY1 is adjusting the responsiveness toward ABA, sequences encoding either the complete WHIRLY1 protein or a truncated form lacking the plastid transit peptide were overexpressed in the why1 mutant background. In plants overexpressing the full-length sequence, WHIRLY1 accumulated in both plastids and the nucleus, whereas in plants overexpressing the truncated sequence, WHIRLY1 accumulated exclusively in the nucleus. Seedlings containing recombinant WHIRLY1 in both compartments were hypersensitive toward ABA. In contrast, seedlings possessing only the nuclear form of WHIRLY1 were as insensitive toward ABA as the why1 mutants. ABA was furthermore shown to lower the rate of germination of wildtype seeds even in the presence of abamine which is known to inhibit the formation of xanthoxin, the plastid located precursor of ABA. From this we conclude that plastid located WHIRLY1 enhances the responsiveness of seeds toward ABA even when ABA is supplied exogenously.

Characterization of a Wheat Heme Oxygenase-1 Gene and Its Responses to Different Abiotic Stresses

In animals and recently in plants, heme oxygenase-1 (HO1) has been found to confer protection against a variety of oxidant-induced cell and tissue injuries. In this study, a wheat (Triticum aestivum) HO1 gene TaHO1 was cloned and sequenced. It encodes a polypeptide of 31.7 kD with a putative N-terminal plastid transit peptide. The amino acid sequence of TaHO1 was found to be 78% similar to that of maize HO1. Phylogenetic analysis revealed that TaHO1 clusters together with the HO1-like sequences in plants. The purified recombinant TaHO1 protein expressed in Escherichia coli was active in the conversion of heme to biliverdin IXa (BV), and showed that the Vmax was 8.8 U·mg−1 protein with an apparent Km value for hemin of 3.04 μM. The optimum Tm and pH were 35 °C and 7.4, respectively. The result of subcellular localization of TaHO1 showed that the putative transit peptide was sufficient for green fluorescent protein (GFP) to localize in chloroplast and implied that TaHO1 gene product is at least localized in the chloroplast. Moreover, we found that TaHO1 mRNA could be differentially induced by the well-known nitric oxide (NO) donor sodium nitroprusside (SNP), gibberellin acid (GA), abscisic acid (ABA), hydrogen peroxide (H2O2) and NaCl treatments. Therefore, the results suggested that TaHO1 might play an important role in abiotic stress responses.

Cloning and characterization of the HLIP gene encoding high light-inducible protein from Porphyra yezoensis

High light-inducible proteins are critical for photosynthetic organisms when responding to high light stress. We cloned and characterized the HLIP gene from Porphyra yezoensis, which was designated as PyHLIP. Sequence analysis revealed that the open reading frame of PyHLIP was 387 bp in length that encoded a 128 amino acid-polypeptide containing a plastid transit peptide and an obvious membrane-spanning α-helix region. No intron existed in the genomic DNA of PyHLIP. The putative promoter of PyHLIP was TATA-less, and the initiator element (Inr) existed upstream of ATG. Analysis of genomic organization indicated there were two copies of PyHLIP gene in the P. yezoensis nuclear genome. The expression of PyHLIP can be induced by various stress conditions and is especially sensitive to high light.

Isolation and Characterization of a Heme Oxygenase-1 Gene from Chinese Cabbage

Heme oxygenase-1 (HO1) is a heme-catabolizing enzyme induced by a variety of stress conditions. This article described the cloning and characterization of BrHO1 gene which codes for a putative HO1 from Chinese cabbage (Brassica rapa subsp. pekinensis). BrHO1 consists of three exons and encodes a protein precursor of 32.3 kD with a putative N-terminal plastid transit peptide. The amino acid sequence of BrHO1 was 84% similar to Arabidopsis counterpart HY1. The three-dimensional structure of BrHO1 showed a high degree of structural conservation compared with the known HO1 crystal structures. Phylogenetic analysis revealed that BrHO1 clearly grouped with the HO1-like sequences. The recombinant BrHO1 protein expressed in Escherichia coli was active in the conversion of heme to biliverdin IXα (BV). Furthermore, the results of subcellular localization of BrHO1 demonstrated that BrHO1 gene product was most likely localized in the chloroplasts. BrHO1 was differently expressed in all tested tissues and could be induced upon osmotic and salinity stresses, cadmium (Cd) exposure, hydrogen peroxide (H(2)O(2)), and hemin treatments. Together, the results suggested that BrHO1 plays an important role in abiotic stress responses.

Genes encoding the biotin carboxylase subunit of acetyl-CoA carboxylase from Brassica napus and parental species: cloning, expression patterns, and evolution

Comparative genomics is a useful tool to investigate gene and genome evolution. Biotin carboxylase (BC), an important subunit of heteromeric acetyl-CoA carboxylase (ACCase) that is a rate-limiting enzyme in fatty acid biosynthesis in dicots, catalyzes ATP, biotin carboxyl carrier protein, and CO2 to form carboxybiotin carboxyl carrier protein. In this study, we cloned four genes encoding BC from Brassica napus L. (namely BnaC.BC.a, BnaC.BC.b, BnaA.BC.a, and BnaA.BC.b), and two were cloned from each of the two parental species Brassica rapa L. (BraA.BC.a and BraA.BC.b) and Brassica oleracea L. (BolC.BC.a and BolC.BC.b). Sequence analyses revealed that in B. napus the genes BnaC.BC.a and BnaC.BC.b were from the C genome of B. oleracea, whereas BnaA.BC.a and BnaA.BC.b were from the A genome of B. rapa. Comparative and cluster analysis indicated that these genes were divided into two major groups, BnaC.BC.a, BnaA.BC.a, BraA.BC.a, and BolC.BC.a in group-1 and BnaC.BC.b, BnaA.BC.b, BraA.BC.b, and BolC.BC.b in group-2. The divergence of group-1 and group-2 genes occurred in their common ancestor 13-17 million years ago (MYA), soon after the divergence of Arabidopsis and Brassica (15-20 MYA). This time of divergence is identical to the previously reported triplicated time of paralogous subgenomes of diploid Brassica species and the divergence date of group-1 and group-2 genes of α-carboxyltransferase, another subunit of heteromeric ACCase, in Brassica. Reverse transcription PCR revealed that the expression level of group-1 and group-2 genes varied in different organs, and the expression patterns of the two groups of genes were similar in different organs, except in flower. However, two paralogs of group-2 BC genes from B. napus could express differently in mature plants tested by generating BnaA.BC.b and BnaC.BC.b promoter-β-glucuronidase (GUS) fusions. The amino acid sequences of proteins encoded by these genes were highly conserved, except the sequence encoding predicted plastid transit peptides. The plastid transit peptides on the BC precursors of Brassica (71-72 amino acid residues) were predicted based on AtBC protein, compared, and confirmed by fusion with green fluorescent protein. Our results will be helpful in elucidating the evolution and the regulation of ACCase in the genus Brassica.

Subcellular localization of rice hexokinase (OsHXK) family members in the mesophyll protoplasts of tobacco

Hexokinase (HXK, EC 2.7.1.1) plays an important role in the metabolism and glucose signalling. To examine the characteristics of HXK gene family in rice, the subcellular localizations of ten hexokinases (OsHXK1 - OsHXK10) were determined using OsHXK::GFP fusion proteins in tobacco mesophyll protoplasts. As was previously demonstrated, OsHXK4 was detected in the chloroplast stroma, OsHXK5 and OsHXK6 in the mitochondria, and OsHXK7 and OsHXK10 in the cytoplasm. In the present study, OsHXKs were clearly divided into three types (A, B, C) based on their N-terminal sequences. The new type-C HXKs in plants, OsHXK1, OsHXK7 and OsHXK8, which lack the plastidic transit peptide and the membrane anchor domain, were detected not only in the cytoplasm but also in the nucleus. The type-B HXKs, OsHXK2, OsHXK3, OsHXK9 and OsHXK10, which contained a membrane anchor domain, were distinctly localized in the mitochondria. These results suggest that OsHXKs localized in different cell compartments may be involved in the glucose signalling-related gene expression during growth and development of rice.

The last gene involved in the MEP pathway of Artemisia annua: Cloning and characterization and functional identification

Hydroxymethylbutenyl 4-diphosphate reductase (HDR) catalyzes the last reaction of the methylerythritol phosphate (MEP) pathway for the biosynthesis of artemisinin precursors, a branching step that separately produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in a ratio of 5:1 to 6:1. The full-length cDNA sequence of HDR was cloned and characterized from Artemisia annua L. for the first time. The new cDNA was designated as AaHDR (GenBank accession No.: GQ119345). The full-length cDNA ofAaHDR was 1640 bp containing a 1368 bp open reading frame (ORF) encoding a polypeptide of 455 amino acids with a calculated molecular mass of 51.3 KDa and an isoelectric point of 5.63. Comparative and bioinformatic analysis revealed that AaHDR had extensive homology with HDRs from other plant species and contained a conserved transit peptide for plastids. The phylogenetic analysis indicated that all HDRs could be divided into three groups and AaHDR belonged to plant HDRs family. Then the homology-based structural modeling of AaHDR showed that AaHDR had the typical structure of HDR fromA. aeolicus, which adopted a cloverleaf or trefoil-like structure with each monomer in the dimer containing three alpha/beta domains surrounding a central [Fe3S4] cluster ligated to Cys13, Cys96 and Cys193. Finally, AaHDR was transformed into the E. coli HDR mutant strain MG1655 ara HDR, which was able to rescue the lethal phenotype of the E. coliHDR mutant strain MG1655 ara-HDR. This confirmed that AaHDR had the typical function of HDR gene. The cloning and characterization of AaHDR will be helpful to understand more about the function of HDR at the level of molecular genetics and unveil the biosynthetic mechanism of artemisinin precursors. The present work also provides a candidate gene for metabolic engineering of the artemisinin biosynthesis pathway in A. annua. Key words: Artemisia annua L., hydroxymethylbutenyl 4-diphosphate reductase, cloning, characterization, functional complementation.

Evolution of plant phage-type RNA polymerases: the genome of the basal angiosperm Nuphar advena encodes two mitochondrial and one plastid phage-type RNA polymerases

BackgroundIn mono- and eudicotyledonous plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type) encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species are limited to the moss Physcomitrella and the spikemoss Selaginella. To further elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP) available, we searched for the respective genes in the basal angiosperm Nuphar advena.ResultsBy screening a set of BAC library filters, three RpoT genes were identified. Both genomic gene sequences and full-length cDNAs were determined. The NaRpoT mRNAs specify putative polypeptides of 996, 990 and 985 amino acids, respectively. All three genes comprise 19 exons and 18 introns, conserved in their positions with those known from RpoT genes of other land plants. The encoded proteins show a high degree of conservation at the amino acid sequence level, including all functional crucial regions and residues known from the phage T7 RNAP. The N-terminal transit peptides of two of the encoded polymerases, NaRpoTm1 and NaRpoTm2, conferred targeting of green fluorescent protein (GFP) exclusively to mitochondria, whereas the third polymerase, NaRpoTp, was targeted to chloroplasts. Remarkably, translation of NaRpoTp mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis and the Nicotiana RpoTp gene, N. advena RpoTp provides another example for a plant mRNA that is exclusively translated from a non-AUG codon. In contrast to the RpoT of the lycophyte Selaginella and those of the moss Physcomitrella, which are according to phylogenetic analyses in sister positions to all other phage-type polymerases of angiosperms, the Nuphar RpoTs clustered with the well separated clades of mitochondrial (NaRpoTm1 and NaRpoTm2) and plastid (NaRpoTp) polymerases.ConclusionsNuphar advena encodes two mitochondrial and one plastid phage-type RNAP. Identification of a plastid-localized phage-type RNAP in this basal angiosperm, orthologous to all other RpoTp enzymes of flowering plants, suggests that the duplication event giving rise to a nuclear gene-encoded plastid RNA polymerase, not present in lycopods, took place after the split of lycopods from all other tracheophytes. A dual-targeted mitochondrial and plastididal RNA polymerase (RpoTmp), as present in eudicots but not monocots, was not detected in Nuphar suggesting that its occurrence is an evolutionary novelty of eudicotyledonous plants like Arabidopsis.

The HDR gene involved in the TIA pathway from Rauvolfia verticillata: Cloning, characterization and functional identification

1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR, EC 1.17.1.2) catalyzes the last reaction of the methylerythritol phosphate (MEP) pathway. The full-length cDNA sequence of HDR was cloned and characterized from terpenoid-indole-alkaloid-producing Rauvolfia verticillata. The new cDNA was named as RvHDR and submitted to GenBank® to be assigned with an accession number: EU034699. The full-length cDNA of RvHDR was 1679-bp containing a 1389-bp open reading frame (ORF) encoding a polypeptide of 462-amino acids with a calculated molecular mass of 52 kDa and an isoelectric point of 5.26. Comparative and bioinformatic analyses revealed that RvHDR had extensive homology with HDRs from other plant species and contained a conserved transit peptide for plastids. The phylogenetic indicated that all HDRs could be divided into three groups and RvHDR belonged to plant HDRs family.RvHDR was found to be expressed in all tested tissues including roots, stems, leaves, flowers and fruits but at different levels. The highest expression level was found in flowers, and higher expression level in leaves and fruits; the expression level was low in roots and lowest in stems. Expression profiling analyses revealed that RvHDR expression was induced by exogenous elicitors including methyl jasmonate, acetyl salicylic acid, abscisic acid and UV, and showed the transcription levels were all up-regulated compared to the control. Finally,RvHDR was transformed into the E. coli HDR mutant strain MG1655 ara HDR, which was able to rescue the lethal phenotype of the E. coli HDR mutant. This confirmed that RvHDR had the typically function of HDR gene. The cloning, characterization and functional identification of RvHDR will be helpful to understand more about the function of HDR at the level of molecular genetics and help to unveil the biosynthetic mechanism of TIAs precursor and provides a candidate gene for metabolic engineering of the TIAs pathway in R. verticillata. Key words: Rauvolfia verticillata, HDR gene, cloning, expression profile, functional complementation.

The sunflower plastidial ω3-fatty acid desaturase (HaFAD7) contains the signalling determinants required for targeting to, and retention in, the endoplasmic reticulum membrane in yeast but requires co-expressed ferredoxin for activity

Although plant plastidial omega3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 omega3-desaturase showed low activity in contrast to similar expression of microsomal FAD3 omega3-desaturases. However, the removal of the plastidial transit peptide and the incorporation of a KKNL motif to the C-terminus of HaFAD7 increased the activity by 10-fold compared to the native protein. N-terminal fusion of transmembrane-domains from either the yeast microsomal ELO3, (a type III signal anchor domain), or FAE1, an endoplasmic reticulum membrane anchoring domain, resulted in moderate increases in enzyme activity (5- and 7-fold, respectively), suggesting that the first, most hydrophobic transmembrane domain of HaFAD7 is sufficient to direct targeting to, and insertion into, the endoplasmic reticulum membrane. Furthermore, fusing a hemagglutinin (HA) epitope tag upstream of an endogenous C-terminal KEK motif resulted in a significant loss of activity compared to the un-tagged construct, indicating that the endogenous KEK C-terminal di-lysine motif is capable of directing in yeast the ER-retention of this normally plastidial-located protein. Western blotting analysis of constructs with internal HA epitope revealed that in whole cell extracts, with the exception of the one bound to C-terminal, it did not display a reduced level of protein accumulation. Whilst ferredoxin was shown to be required for HaFAD7 activity in yeast, it appears not necessary for protein stability and accumulation of this plastidial desaturase in the endoplasmic reticulum.

The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from Taxus media: Cloning, characterization and functional identification

1-Hydroxy-2-methyl-butenyl 4-diphosphate reductase (HDR:EC: 1.17.1.2) is the last and key enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, which provides the general precursors for taxol biosynthesis and is the important candidate gene for metabolic engineering of the taxol biosynthetic pathway. The gene encoding HDR from Taxus media (designated as TmHDR; GenBank accession number: EF541129) was cloned using RACE, characterized at the levels of bioinformatics and tissue expression pattern, and finally functionally identified with the genetic complementation. The full-length cDNA of TmHDR was 1987 bp containing an 1425-bp open reading frame (ORF) that encoded a polypeptide of 474 amino acids with a calculated molecular mass of 53.2 kDa and an isoelectric point of 5.65. Comparative and bioinformatic analysis revealed that TmHDR showed extensive homology with HDRs from other plant species. The subcellular prediction showed that TmHDR owned a plastidial transit peptide of 47 amino acids at its N terminus, which directed TmHDR to plastid. The phylogenetic analysis revealed that TmHDR belonged to gymnosperm HDRs. The tissue expression pattern analysis indicated that TmHDR expressed in all tested tissues including cortices, stems, roots and leaves but at different levels. The highest expression level of TmHDR was found in leaves, followed by roots and cortices; and the expression of TmHDR was very low in stems. Finally, TmHDR was functionally expressed in lethal E. coli HDR mutant and could rescue the mutants. So, the genetic complementation assay demonstrated that TmHDR did encode the protein that had the typical activity of HDR proteins. In summary, the present study will be helpful to understand more about the role of HDR involved in the Taxol biosynthesis at the molecular level and provides an important candidate gene for metabolic engineering of the taxol biosynthesis in Taxus species plants. Key words: 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase (HDR), cloning, genetic complementation, Taxus media, tissue expression.