N-terminal Transit Peptide Research Articles

The C-terminus of Bienertia sinuspersici Toc159 contains essential elements for its targeting and anchorage to the chloroplast outer membrane.

Most nucleus-encoded chloroplast proteins rely on an N-terminal transit peptide (TP) as a post-translational sorting signal for directing them to the organelle. Although Toc159 is known to be a receptor for specific preprotein TPs at the chloroplast surface, the mechanism for its own targeting and integration into the chloroplast outer membrane is not completely understood. In a previous study, we identified a novel TP-like sorting signal at the C-terminus (CT) of a Toc159 homolog from the single-cell C4 species, Bienertia sinuspersici. In the current study, we have extended our understanding of the sorting signal using transient expression of fluorescently-tagged fusion proteins of variable-length, and with truncated and swapped versions of the CT. As was shown in the earlier study, the 56 residues of the CT contain crucial sorting information for reversible interaction of the receptor with the chloroplast envelope. Extension of this region to 100 residues in the current study stabilized the interaction via membrane integration, as demonstrated by more prominent plastid-associated signals and resistance of the fusion protein to alkaline extraction. Despite a high degree of sequence similarity, the plastid localization signals of the equivalent CT regions of Arabidopsis thaliana Toc159 homologs were not as strong as that of the B. sinuspersici counterparts. Together with computational and circular dichroism analyses of the CT domain structures, our data provide insights into the critical elements of the CT for the efficient targeting and anchorage of Toc159 receptors to the dimorphic chloroplasts in the single-cell C4 species.

Unexpected roles for ancient proteins: flavone 8-hydroxylase in sweet basil trichomes is a Rieske-type, PAO-family oxygenase.

Most elucidated hydroxylations in plant secondary metabolism are catalyzed by oxoglutarate- or cytochrome P450-dependent oxygenases. Numerous hydroxylations still evade clarification, suggesting that they might be performed by alternative enzyme types. Here, we report the identification of the flavone 8-hydroxylase (F8H) in sweet basil (Ocimum basilicum L.) trichomes as a Rieske-type oxygenase. Several features of the F8H activity in trichome protein extracts helped to differentiate it from a cytochrome P450-catalyzed reaction and identify candidate genes in the basil trichome EST database. The encoded ObF8H proteins share approximately 50% identity with Rieske-type protochlorophyllide a oxygenases (PTC52) from higher plants. Homology cloning and DNA blotting revealed the presence of several PTC52-like genes in the basil genome. The transcripts of the candidate gene designated ObF8H-1 are strongly enriched in trichomes compared to whole young leaves, indicating trichome-specific expression. The full-length ObF8H-1 protein possesses a predicted N-terminal transit peptide, which directs green fluorescent protein at least in part to chloroplasts. The F8H activity in crude trichome protein extracts correlates well with the abundance of ObF8H peptides. The purified recombinant ObF8H-1 displays high affinity for salvigenin and is inactive with other tested flavones except cirsimaritin, which is 8-hydroxylated with less than 0.2% relative activity. The efficiency of in vivo 8-hydroxylation by engineered yeast was improved by manipulation of protein subcellular targeting. blast searches showed that occurrence of several PTC52-like genes is rather common in sequenced plant genomes. The discovery of ObF8H suggests that Rieske-type oxygenases may represent overlooked candidate catalysts for oxygenations in specialized plant metabolism.

Chloroplast-encoded serotonin N-acetyltransferase in the red alga Pyropia yezoensis: gene transition to the nucleus from chloroplasts.

Melatonin biosynthesis involves the N-acetylation of arylalkylamines such as serotonin, which is catalysed by serotonin N-acetyltransferase (SNAT), the penultimate enzyme of melatonin biosynthesis in both animals and plants. Here, we report the functional characterization of a putative N-acetyltransferase gene in the chloroplast genome of the alga laver (Pyropia yezoensis, formerly known as Porphyra yezoensis) with homology to the rice SNAT gene. To confirm that the putative Pyropia yezoensis SNAT (PySNAT) gene encodes an SNAT, we cloned the full-length chloroplastidic PySNAT gene by PCR and purified the recombinant PySNAT protein from Escherichia coli. PySNAT was 174 aa and had 50% amino acid identity with cyanobacteria SNAT. Purified recombinant PySNAT showed a peak activity at 55 °C with a K m of 467 µM and V max of 28 nmol min-1 mg(-1) of protein. Unlike other plant SNATs, PySNAT localized to the cytoplasm due to a lack of N-terminal chloroplast transit peptides. Melatonin was present at 0.16ng g(-1) of fresh mass but increased during heat stress. Phylogenetic analysis of the sequence suggested that PySNAT has evolved from the cyanobacteria SNAT gene via endosymbiotic gene transfer. Additionally, the chloroplast transit peptides of plant SNATs were acquired 1500 million years ago, concurrent with the appearance of green algae.

Translational fusion and redirection to thylakoid lumen as strategies to enhance accumulation of human papillomavirus E7 antigen in tobacco chloroplasts.

Human papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death in women worldwide, and its E7 antigen is the major candidate for a therapeutic vaccine. The large scale production of E7 by molecular farming that would lead to the development of a safe and inexpensive vaccine is impaired by its low accumulation level in the plant cell. To enhance antigen production in the plastids, two alternative strategies were carried out: the expression of E7 as a translational fusion to β-glucuronidase enzyme and redirection of E7 into the thylakoid lumen. The use of the β-glucuronidase as a partner protein turned out to be a successful strategy, antigen expression levels were enhanced between 30 and 40 times relative to unfused E7. Moreover, best accumulation, albeit at a high metabolic cost that compromised biomass production, was obtained redirecting E7 into the thylakoid lumen by the incorporation of the N-terminal transit peptide, Str. Following this approach lumenal E7 production exceeded the stromal by two orders of magnitude. Our results highlight the relevance of exploring different strategies to improve recombinant protein stability for certain transgenes in order to exploit potential advantages of recombinant protein accumulation in chloroplasts.

A new member of the psToc159 family contributes to distinct protein targeting pathways in pea chloroplasts.

Protein import into chloroplasts relies on specific targeting of preproteins from the cytosol to the organelles and coordinated translocation processes across the double envelope membrane. Here, two complex machineries constitute the so called general import pathway, which consists of the TOC and TIC complexes (translocon at the outer envelope of chloroplasts and translocon at the inner envelope of chloroplasts, respectively). The majority of canonical preproteins feature an N-terminal cleavable transit peptide, which is necessary for targeting and recognition at the chloroplast surface by receptors of TOC, where Toc159 acts as the primary contact site. We identified a non-canonical preprotein without the classical transit peptide, the superoxide dismutase (FSD1), which was then used in chemical crosslinking approaches to find new interaction partners at the outer envelope from pea chloroplasts. In this way we could link FSD1 to members of the Toc159 family in pea, namely psToc132 and psToc120. Using deletion mutants as well as a peptide scanning approach we defined regions of the preprotein, which are involved in receptor binding. These are distributed across the entire sequence; however the extreme N-terminus as well as a C-proximal domain turned out to be essential for targeting and import. En route into the plastid FSD1 engages components of the general import pathway, implying that in spite of the non-canonical targeting information and recognition by a specific receptor this preprotein follows a similar way across the envelope as the majority of plastid preproteins.

FLOURY ENDOSPERM6 encodes a CBM48 domain‐containing protein involved in compound granule formation and starch synthesis in rice endosperm

Starch is the most widespread form of energy storage in the plant kingdom. Although many enzymes and related factors have been identified for starch biosynthesis, unknown players remain to be identified, given that it is a complicated and sophisticated process. The endosperm of rice (Oryza sativa) has been used for the study of starch synthesis. Here, we report the cloning and characterization of the FLOURY ENDOSPERM6 (FLO6) gene in rice. In the flo6 mutant, the starch content is decreased and the normal physicochemical features of starch are changed. Significantly, flo6 mutant endosperm cells show obvious defects in compound granule formation. Map-based cloning showed that FLO6 encodes a protein of unknown function. It harbors an N-terminal transit peptide that ensures its correct localization and functions in the plastid, and a C-terminal carbohydrate-binding module 48 (CBM48) domain that binds to starch. Furthermore, FLO6 can interact with isoamylase1 (ISA1) both in vitro and in vivo, whereas ISA1 does not bind to starch directly. We thus propose that FLO6 may act as a starch-binding protein involved in starch synthesis and compound granule formation through a direct interaction with ISA1 in developing rice seeds. Our data provide a novel insight into the role of proteins with the CBM48 domain in plant species.

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.

A Mutable Albino Allele in Rice Reveals that Formation of Thylakoid Membranes Requires the SNOW-WHITE LEAF1 Gene

Active DNA transposons are important tools for gene functional analysis. The endogenous non-autonomous transposon, nDart1-0, in rice (Oryza sativa L.) is expected to generate various transposon-insertion mutants because nDart1-0 elements tend to insert into genic regions under natural growth conditions. We have developed a specific method (nDart1-0-iPCR) for efficient detection of nDart1-0 insertions and successfully identified the SNOW-WHITE LEAF1 (SWL1) gene in a variegated albino (swl1-v) mutant obtained from the nDart1-promoted rice tagging line. The variegated albino phenotype was caused by insertion and excision of nDart1-0 in the 5'-untranslated region of the SWL1 gene predicted to encode an unknown protein with the N-terminal chloroplast transit peptide. SWL1 expression was detected in various rice tissues at different developmental stages. However, immunoblot analysis indicated that SWL1 protein accumulation was strictly regulated in a tissue-specific manner. In the swl1 mutant, formations of grana and stroma thylakoids and prolamellar bodies were inhibited. This study revealed that SWL1 is essential for the beginning of thylakoid membrane organization during chloroplast development. Furthermore, we provide a developmental perspective on the nDart1-promoted tagging line to characterize unidentified gene functions in rice.

Seedling Lethal1, a Pentatricopeptide Repeat Protein Lacking an E/E+ or DYW Domain in Arabidopsis, Is Involved in Plastid Gene Expression and Early Chloroplast Development

Chloroplasts are the site of photosynthesis and the biosynthesis of essential metabolites, including amino acids, fatty acids, and secondary metabolites. It is known that many seedling-lethal mutants are impaired in chloroplast function or development, indicating the development of functional chloroplast is essential for plant growth and development. Here, we isolated a novel transfer DNA insertion mutant, dubbed sel1 (for seedling lethal1), that exhibited a pigment-defective and seedling-lethal phenotype with a disrupted pentatricopeptide repeat (PPR) gene. Sequence analysis revealed that SEL1 is a member of the PLS subgroup, which is lacking known E/E(+) or DYW domains at the C terminus, in the PLS subfamily of the PPR protein family containing a putative N-terminal transit peptide and 14 putative PPR or PPR-like motifs. Confocal microscopic analysis showed that the SEL1-green fluorescent protein fusion protein is localized in chloroplasts. Transmission electron microscopic analysis revealed that the sel1 mutant is impaired in the etioplast, as well as in chloroplast development. In sel1 mutants, plastid-encoded proteins involved in photosynthesis were rarely detected due to the lack of the corresponding transcripts. Furthermore, transcript profiles of plastid genes revealed that, in sel1 mutants, the transcript levels of plastid-encoded RNA polymerase-dependent genes were greatly reduced, but those of nuclear-encoded RNA polymerase-dependent genes were increased or not changed. Additionally, the RNA editing of two editing sites of the acetyl-CoA carboxylase beta subunit gene transcripts in the sel1 mutant was compromised, though it is not directly connected with the sel1 mutant phenotype. Our results demonstrate that SEL1 is involved in the regulation of plastid gene expression required for normal chloroplast development.

Transit peptide design and plastid import regulation

Import of most nuclear encoded proteins into plastids is directed by an N-terminal transit peptide. Early studies suggested that transit peptides are interchangeable between precursor proteins. However, emerging evidence shows that different transit peptides contain different motifs specifying their preference for certain plastid types or ages. In this opinion article, we propose a 'multi-selection and multi-order' (M&M) model for transit peptide design, describing each transit peptide as an assembly of motifs for interacting with selected translocon components. These interactions determine the preference of the precursor for a particular plastid type or age. Furthermore, the order of the motifs varies among transit peptides, explaining why no consensus sequences have been identified through linear sequence comparison of all transit peptides as one group.

Molecular cloning and characterization of the gene encoding 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase from hairy roots of Rauvolfia verticillata

Abstract2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (MCT) catalyzes the third reaction in the plastidial non-mevalonate pathway, which provides the precursors for ajmalicine. A full-length cDNA encoding MCT (RvMCT) was identified from hairy roots of Rauvolfia verticillata. The full-length 1,499-bp cDNA of RvMCT had a 945-bp coding sequence that encoded a 314-amino-acid protein with an N-terminal chloroplast transit peptide of 67 amino acid residues. RvMCT exhibited homology with other plant MCTs at the levels of sequence and structure. The phylogenetic analysis revealed the plant MCTs could be divided into three separated clusters including gymnosperms, monocotyledons and dicotyledons. Gene expression of ajmalicine metabolism (DXR, MCT, MECS, HDS, HDR, STR and SGD) in hairy roots, roots, stems, old leaves, young leaves and barks was analyzed by quantitative PCR. All the seven genes had higher expression levels in hairy roots than in other plant organs. This suggested hairy roots of R. verticillata possessed more active alkaloid metabolism than other organs and it was the reason that hairy roots produced higher levels of ajmalicine. Furthermore, the expression of DXR, MECS, HDS, HDR, STR and SGD genes was not detected in stems (only MCT detected in stems), so it could be presumed that stem acted as a transporter tissue of ajmalicine. Finally, the colour complementation assay indicated that the function of RvMCT was the same as Arabidopsis MCT. Molecular cloning, characterization and functional identification of RvMCT will be helpful to understand more about the role of MCT involved in ajmalicine biosynthesis at the molecular level.

Molecular cloning and sequence comparison of a cDNA encoding α-glucan, water dikinase (GWD) from potato (Solanum tuberosum L.), and analysis of gene expression

Starch phosphorylation is an important biochemical aspect of plant starch metabolism as it influences the overall structure of the starch granule, and a prerequisite for its degradation. There is a growing interest on the isolation and characterization of α-glucan/glucan-like, water dikinases (GWDs) from plants, particularly agriculturally important crops, because GWD is known to catalyze starch phosphorylation both in leaves and different plant storage organs. In the present study, a 4,789-bp full-length cDNA encoding a GWD isoform was isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR approach using tuber RNA. The predicted protein consisted of 1,463 amino acids having N-terminal 77-amino acid transit peptide, and 1,386-amino acid mature protein shorter by one amino acid as compared to the other mature GWDs from potato and tomato. The mature GWD showed 98 % sequence identity with the GWD isolated earlier from the potato cv. Desiree. Variations were found at 25 locations representing mostly non-conservative substitutions. The GWD represents a distinct isoform from potato, as revealed by sequence and phylogenetic analyses. Amino acid composition, segment-wise hydrophobic characters, predicted secondary structures were also analyzed and documented in this report. Broadly, the level of GWD expression as analyzed by semi-quantitative RT-PCR approach was found to be nearly uniform both in the mature tubers and leaves from most of the potato cultivars. By immunodetection technique, a band corresponding to ~155 kDa protein was detected only in the tuber protein extracts. The tuber starch-bound phosphorus content data showed minor variations between the potato cultivars.

Disrupting the bimolecular binding of the haem-binding protein 5 (AtHBP5) to haem oxygenase 1 (HY1) leads to oxidative stress in Arabidopsis

The Arabidopsis thaliana L. SOUL/haem-binding proteins, AtHBPs belong to a family of five members. The Arabidopsis cytosolic AtHBP1 (At1g17100) and AtHBP2 (At2g37970) have been shown to bind porphyrins and metalloporphyrins including haem. In contrast to the cytosolic localization of these haem-binding proteins, AtHBP5 (At5g20140) encodes a protein with an N-terminal transit peptide that probably directs targeting to the chloroplast. In this report, it is shown that AtHBP5 binds haem and interacts with the haem oxygenase, HY1, in both yeast two-hybrid and BiFC assays. The expression of HY1 is repressed in the athbp5 T-DNA knockdown mutant and the accumulation of H2O2 is observed in athbp5 seedlings that are treated with methyl jasmonate (MeJA), a ROS-producing stress hormone. In contrast, AtHBP5 over-expressing plants show a decreased accumulation of H2O2 after MeJA treatment compared with the controls. It is proposed that the interaction between the HY1 and AtHBP5 proteins participate in an antioxidant pathway that might be mediated by reaction products of haem catabolism.

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.

A novel rhodanese is required to maintain chloroplast translation in Chlamydomonas

Rhodanese-domain proteins (RDPs) are widespread in plants and other organisms, but their biological roles are mostly unknown. Here we report on a novel RDP from Chlamydomonas that has a single rhodanese domain, and a predicted chloroplast transit peptide. The protein was produced in Escherichia coli with a His-tag, but lacking most of the N-terminal transit peptide, and after purification was found to have rhodanese activity in vitro. It was also used to elicit antibodies for western blot analysis, which showed that the native Chlamydomonas protein migrated slower on SDS gels (apparent M(r) =34 kDa) than its predicted size (27 kDa), and co-fractionated with chloroplasts. To assess function in vivo, the tandem-RNAi approach was used to generate Chlamydomonas strains that had reductions of 30-70% for the mRNA and ~20-40% for the 34-kDa protein. These strains showed reduced growth under all trophic conditions, and were sensitive to even moderate light; properties reminiscent of chloroplast translation mutants. Pulse-labeling in the presence of cycloheximide indicated that chloroplast protein synthesis was broadly reduced in the RNAi strains, and transcript analysis (by RT-PCR and northern blotting) indicated the effect was mainly translational. These results identify a novel rhodanese-like protein that we have named CRLT, because it is required to maintain chloroplast translation.

A single gene encodes isopentenyl diphosphate isomerase isoforms targeted to plastids, mitochondria and peroxisomes in Catharanthus roseus

Isopentenyl diphosphate isomerases (IDI) catalyze the interconversion of the two isoprenoid universal C5 units, isopentenyl diphosphate and dimethylally diphosphate, to allow the biosynthesis of the large variety of isoprenoids including both primary and specialized metabolites. This isomerisation is usually performed by two distinct IDI isoforms located either in plastids/peroxisomes or mitochondria/peroxisomes as recently established in Arabidopsis thaliana mainly accumulating primary isoprenoids. By contrast, almost nothing is known in plants accumulating specialized isoprenoids. Here we report the cloning and functional validation of an IDI encoding cDNA (CrIDI1) from Catharanthus roseus that produces high amount of monoterpenoid indole alkaloids. The corresponding gene is expressed in all organs including roots, flowers and young leaves where transcripts have been detected in internal phloem parenchyma and epidermis. The CrIDI1 gene also produces long and short transcripts giving rise to corresponding proteins with and without a N-terminal transit peptide (TP), respectively. Expression of green fluorescent protein fusions revealed that the long isoform is targeted to both plastids and mitochondria with an apparent similar efficiency. Deletion/fusion experiments established that the first 18-residues of the N-terminal TP are solely responsible of the mitochondria targeting while the entire 77-residue long TP is needed for an additional plastid localization. The short isoform is targeted to peroxisomes in agreement with the presence of peroxisome targeting sequence at its C-terminal end. This complex plastid/mitochondria/peroxisomes triple targeting occurring in C. roseus producing specialized isoprenoid secondary metabolites is somehow different from the situation observed in A. thaliana mainly producing housekeeping isoprenoid metabolites.

ApicoAP: The First Computational Model for Identifying Apicoplast-Targeted Proteins in Multiple Species of Apicomplexa

BackgroundMost of the parasites of the phylum Apicomplexa contain a relict prokaryotic-derived plastid called the apicoplast. This organelle is important not only for the survival of the parasite, but its unique properties make it an ideal drug target. The majority of apicoplast-associated proteins are nuclear encoded and targeted post-translationally to the organellar lumen via a bipartite signaling mechanism that requires an N-terminal signal and transit peptide (TP). Attempts to define a consensus motif that universally identifies apicoplast TPs have failed.Methodology/Principal FindingsIn this study, we propose a generalized rule-based classification model to identify apicoplast-targeted proteins (ApicoTPs) that use a bipartite signaling mechanism. Given a training set specific to an organism, this model, called ApicoAP, incorporates a procedure based on a genetic algorithm to tailor a discriminating rule that exploits the known characteristics of ApicoTPs. Performance of ApicoAP is evaluated for four labeled datasets of Plasmodium falciparum, Plasmodium yoelii, Babesia bovis, and Toxoplasma gondii proteins. ApicoAP improves the classification accuracy of the published dataset for P. falciparum to 94%, originally 90% using PlasmoAP.Conclusions/SignificanceWe present a parametric model for ApicoTPs and a procedure to optimize the model parameters for a given training set. A major asset of this model is that it is customizable to different parasite genomes. The ApicoAP prediction software is available at http://code.google.com/p/apicoap/ and http://bcb.eecs.wsu.edu.

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...

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 a Heme Oxygenase-2 Gene from Alfalfa (Medicago sativa L.)

Heme oxygenase (HO, EC 1.14.99.3) catalyzes the oxidation of heme and performs vital roles in plant development and stress responses. Two HO isozymes exist in plants. Between these, HO-1 is an oxidative stress-response protein, and HO-2 usually exhibited constitutive expression. Although alfalfa HO-1 gene (MsHO1) has been investigated previously, HO2 is still poorly understood. In this study, we report the cloning and characterization of HO2 gene, MsHO2, from alfalfa (Medica sativa L.). The full-length cDNA of MsHO2 contains an ORF of 870 bp and encodes for 290 amino acid residues with a predicted molecular mass of 33.3 kDa. Similar to MsHO1, MsHO2 also appears to have an N-terminal transit peptide sequence for chloroplast import. Many conserved residues in plant HO were also conserved in MsHO2. However, unlike HO-1, the conserved histidine (His) required for heme-iron binding and HO activity was replaced by tyrosine (Tyr) in MsHO2. Further biochemical activity analysis of purified mature MsHO2 showed no HO activity, suggesting that MsHO2 may not be a true HO in nature. Semi-quantitative RT-PCR confirmed its maximum expression in the germinating seeds. Importantly, the expression levels of MsHO2 were up-regulated under sodium nitroprusside (SNP) and H(2)O(2) (especially) treatment, respectively.