Correlation between the induction of a gene for Δ1-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress

The isolation and characterization is reported of a cDNA for Δ¹-pyrroline-5-carboxylate (P5C) synthetase (cAtP5CS), an enzyme involved in the biosynthesis of proline, from a cDNA library prepared from a dehydrated rosette plant of Arabidopsis thaliana . Southern blot analysis suggested that only one copy of the corresponding gene ( AtP5CS ) is present in A. thaliana . The deduced amino acid sequence of the P5CS protein (AtP5CS) from A. thaliana exhibited 74% homology to that of the P5CS from Vigna aconitifolia . Northern blot analysis revealed that the gene for P5CS was induced by dehydration, high salt and treatment with ABA, while it was not induced by heat or cold treatment. Moreover, the simultaneous accumulation of proline was observed as a result of the former treatments in A. thaliana . A cDNA for P5C reductase (cAtP5CR) was also isolated from A. thaliana and Northern blot analysis was performed. The AtP5CR gene was not induced to a significant extent by dehydration or high-salt stress. These observations suggest that the AtP5CS gene plays a principal role in the biosynthesis of proline in A. thaliana under osmotic stress.     

Molecular cloning and characterization of a cDNA encoding proline transporter in rice

A cDNA encoding a proline (Pro) transporter (ProT) was isolated and characterized from a cDNA library prepared from 14-d-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the rice ProT protein (OsProT) had 68.8% homology to the ProT protein 1 from Arabidopsis thaliana and 59.6% homology to that from Lycopersicon esculentum. Northern blot analysis revealed that the gene for OsProT (OsProT) was expressed in all organs examined, comparatively strongly in leaf sheath and stem. Salt treatment did not induce expression of OsProT but strongly induced expression of the gene for delta1-pyrroline-5-carboxylate synthetase (P5CS), a key enzyme in Pro biosynthesis. Southern blot analysis revealed that OsProT has a gene family. OsProT specifically transported L-Pro in a transport assay using Xenopus laevis oocytes.     

Molecular cloning, expression and mapping analysis of a novel cytosolic ascorbate peroxidase gene from tomato.

Ascorbate peroxidase (APX, EC 1.11.1.11) plays a major role in H(2)O(2)-scavenging in plants and can help to avoid reactive oxygen species (ROS) damage. A new cytosolic APX gene was cloned from tomato (designated LecAPX2) by RACE-PCR. The full-length cDNA of LecAPX2 contained a complete open reading frame (ORF) of 753 bp, which encoding 250 amino acid residues. Homology analysis of LecAPX2 showed a 94% identity with potato cAPX gene and 92% identity with another tomato cAPX gene (APX20), the deduced amino acid showed 88% homology with APX20 protein and 75-92% identity with cAPX from other plants such as potato, tobacco, broccoli, spinach, pea, rice, etc. LecAPX2 revealed the existence of a haem peroxidase and plant APX family signatures. Northern blot analysis showed that LecAPX2 was constitutively expressed in root, stem, leaf, flower and fruit of tomato, whereas the expression levels were different. LecAPX2 was mapped to 6-A using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii.     

A cDNA clone for a pathogenesis-related protein 1 from barley

A barley cDNA clone (PRb-1) corresponding to an mRNA differentially induced in resistant compared to susceptible barley cultivars by powdery mildew infection was isolated and characterised. The deduced amino acid sequence revealed 24 amino acids comprising the signal peptide and 140 amino acids of the mature peptide (15 kDa). This showed close homology to PR-1-like proteins, which have been isolated from maize, tobacco, tomato and Arabidopsis thaliana. Northern blot analysis showed accumulation of the corresponding mRNA 12 h after inoculation of resistant barley cultivars with Erysiphe graminis. Increased expression of the PRb-1 gene was also observed in resistant compared with near-isogenic susceptible barley plants following treatment with ethylene, salicylic acid, methyl jasmonate and 2,6-dichloro-isonicotinic acid.     

A small GTP-binding protein from Arabidopsis thaliana functionally complements the yeast YPT6 null mutant.

A clone designated A.t.RAB6 encoding a small GTP-binding protein was isolated from a cDNA library of Arabidopsis thaliana leaf tissue. The predicted amino acid sequence was highly homologous to the mammalian and yeast counterparts, H.Rab6 and Ryh1/Ypt6, respectively. Lesser homology was found between the predicted Arabidopsis protein sequence and two small GTP-binding proteins isolated from plant species (44% homology to Zea mays Ypt1 and 43% homology to Nicotiana tabacum Rab5). Conserved stretches in the deduced amino acid sequence of A.t.Rab6 include four regions involved in GTP-binding, an effector region, and C-terminal cysteine residues required for prenylation and subsequent membrane attachment. Northern blot analysis demonstrated that A.t.Rab6 mRNA was expressed in root, leaf, stem, and flower tissues from A. thaliana with the highest levels present in roots. Escherichia coli produced histidine-tagged A.t.Rab6 protein-bound GTP, whereas a mutation in one of the guanine nucleotide-binding sites (asparagine122 to isoleucine) rendered it incapable of binding GTP. Functionally, the A.t.RAB6 gene was able to complement the temperature-sensitive phenotype of the YPT6 null mutant in yeast. The isolation of this gene will aid in the dissection of the machinery involved in soluble protein sorting at the trans-Golgi network of plants.     

Characterization of the gene for Δ1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L.

A cDNA for ¹-pyrroline-5-carboxylate (P5C) synthetase (cOsP5CS), an enzyme involved in the biosynthesis of proline, was isolated and characterized from a cDNA library prepared from 14-day-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the P5CS protein (OsP5CS) from O. sativa exhibited 74.2% and 75.5% homology to that of the P5CS from Arabidopsis thaliana and Vigna aconitifolia, respectively. Northern blot analysis revealed that the gene for P5CS (OsP5CS) was induced by high salt, dehydration, treatment of ABA and cold treatment, while it was not induced by heat treatment. Simultaneously, accumulation of proline was observed as a result of high salt treatment in O. sativa. Moreover, the levels of expression of OsP5CS mRNA and content of proline under salt stress condition were compared between a salt-tolerant cultivar, Dee-gee-woo-gen (DGWG) and a salt-sensitive breeding line, IR28. It was observed that the expression of the P5CS gene and the accumulation of proline in DGWG steadily increased, whereas those in IR28 increased slightly.     

A root-specific O-methyltransferase gene expressed in salt-tolerant barley

A cDNA encoding an O-methyltransferase (OMT) was isolated from salt-tolerant barley roots by subtraction hybridization with cDNAs of salt-tolerant barley roots as a tester cDNA and cDNAs of the salt-sensitive barley roots as a driver cDNA. The deduced amino acid sequence showed significant identity with plant caffeic acid/5-hydroxyferulic acid OMTs. Southern blot analysis showed that the OMT gene was a single copy in both salt-tolerant and -sensitive barley. The cloned gene was expressed in a wheat germ cell-free system to produce the OMT, which had methylating activity for caffeic acid. Northern blot analysis showed that the OMT gene was expressed constitutively in the salt-tolerant barley roots and the expression level was increased 1.5 times by salt stress, but the salt-sensitive barley showed no expression of the gene in roots and leaves.     

Molecular cloning and expression characteristics of alternative oxidase gene of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

A novel alternative oxidase (AOX) gene derived from cotton (Gossypium hirsutum), designated as GhAOX1, was cloned with RACE-PCR. The full-length cDNA of GhAOX1was 1,298 bp in size, containing a 996 bp open reading frame (ORF) which corresponds to a precursor protein of 332 amino acid residues with a calculated molecular mass of 37.5 kDa. The predicted amino acid sequence exhibited 68.4%, 68.1%, 59.4%, and 69.8% homology to the alternative oxidases of Arabidopsis thaliana, Nicotiana tabacum, Solanum tuberosum and Glycine max, respectively. Interestingly, striking similarity in several coding regions, such as metal binding and hydrophobic alpha-helix regions was seen between GhAOX1 and other AOX1 proteins. Analysis of the exon/intron structure of the GhAOX1 gene showed that GhAOX1 consisted of four exons and three introns. Southern analysis indicated that the GhAOX1 was a single copy gene belonging to a multi-gene family. Expression analysis by Northern blot revealed that the GhAOX1 exhibited preferential expression in tissues, with the higher expression being found in cotyledons and petals. GhAOX1 was also found to be induced by a variety of stresses stimulation including cold, citrate, SA, KCN and antimycin A in cotton.     

Development of 5006 Full-Length CDNAs in Barley: A Tool for Accessing Cereal Genomics Resources

A collection of 5006 full-length (FL) cDNA sequences was developed in barley. Fifteen mRNA samples from various organs and treatments were pooled to develop a cDNA library using the CAP trapper method. More than 60% of the clones were confirmed to have complete coding sequences, based on comparison with rice amino acid and UniProt sequences. Blastn homologies (E<1E-5) to rice genes and Arabidopsis genes were 89 and 47%, respectively. Of the 5028 possible amino acid sequences derived from the 5006 FLcDNAs, 4032 (80.2%) were classified into 1678 GreenPhyl multigenic families. There were 555 cDNAs showing low homology to both rice and Arabidopsis. Gene ontology annotation by InterProScan indicated that many of these cDNAs (71%) have no known molecular functions and may be unique to barley. The cDNAs showed high homology to Barley 1 GeneChip oligo probes (81%) and the wheat gene index (84%). The high homology between FLcDNAs (27%) and mapped barley expressed sequence tag enabled assigning linkage map positions to 151–233 FLcDNAs on each of the seven barley chromosomes. These comprehensive barley FLcDNAs provide strong platform to connect pre-existing genomic and genetic resources and accelerate gene identification and genome analysis in barley and related species.Key words: full-length cDNA, Hordeum vulgare, mRNA, gene ontology     

The sorting signals for peroxisomal membrane-bound ascorbate peroxidase are within its C-terminal tail

Peroxisomal ascorbate peroxidase (APX) is a carboxyl tail-anchored, type II (N(cytosol)-C(matrix)) integral membrane protein that functions in the regeneration of NAD(+) in glyoxysomes of germinated oilseeds and protection of peroxisomes in other organisms from toxic H(2)O(2). Recently we showed that cottonseed peroxisomal APX was sorted post-translationally from the cytosol to peroxisomes via a novel reticular/circular membranous network that was interpreted to be a subdomain of the endoplasmic reticulum (ER), named peroxisomal ER (pER). Here we report on the molecular signals responsible for sorting peroxisomal APX. Deletions or site-specific substitutions of certain amino acid residues within the hydrophilic C-terminal-most eight-amino acid residues (includes a positively charged domain found in most peroxisomal integral membrane-destined proteins) abolished sorting of peroxisomal APX to peroxisomes via pER. However, the C-terminal tail was not sufficient for sorting chloramphenicol acetyltransferase to peroxisomes via pER, whereas the peptide plus most of the immediately adjacent 21-amino acid transmembrane domain (TMD) of peroxisomal APX was sufficient for sorting. Replacement of the peroxisomal APX TMD with an artificial TMD (devoid of putative sorting sequences) plus the peroxisomal APX C-terminal tail also sorted chloramphenicol acetyltransferase to peroxisomes via pER, indicating that the peroxisomal APX TMD does not possess essential sorting information. Instead, the TMD appears to confer the proper context required for the conserved positively charged domain to function within peroxisomal APX as an overlapping pER sorting signal and a membrane peroxisome targeting signal type 2.     

An oxidative and salinity stress induced peroxisomal ascorbate peroxidase from Avicennia marina: Molecular and functional characterization

APX (EC, 1.11.1.11) has a key role in scavenging ROS and in protecting cells against their toxic effects in algae and higher plants. A cDNA encoding a peroxisomal ascorbate peroxidase, Am-pAPX1, was isolated from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening and its expression in the context of various environmental stresses was investigated. Am-pAPX1 contains an ORF of 286 amino acids coding for a 31.4kDa protein. The C-terminal region of the Am-pAPX1 ORF has a putative transmembrane domain and a peroxisomal targeting signal (RKKMK), suggesting peroxisomal localization. The peroxisomal localization of Am-pAPX1 was confirmed by stable transformation of the GFP-(Ala)(10)-Am-pAPX1 fusion in tobacco. RNA blot analysis revealed that Am-pAPX1 is expressed in response to salinity (NaCl) and oxidative stress (high intensity light, hydrogen peroxide application and excess iron). The isolated genomic clone of Am-pAPX1 was found to contain nine exons. A fragment of 1616bp corresponding to the 5' upstream region of Am-pAPX1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative light and abiotic stress regulatory elements.