Sequence variability of nine cytosolic ascorbate peroxidases in polyploid strawberry.

Little is known about differential gene expression at the molecular level in polyploid plants. Here, we describe the molecular analysis of ApxSC (cytosolic ascorbate peroxidase from a polyploid strawberry) genes. Fifty-three cDNAs encoding ApxSC were isolated from a strawberry fruit cDNA library. These clones were categorized (i) into nine homologous (95 to 99%) gene groups on the basis of their nucleotide sequences and (ii) into four groups of similar (> 98%) polypeptides on the basis of their deduced amino acid sequences. Sequence variation among the gene groups was dispersed throughout the gene, while differences among the polypeptide groups were observed only at three amino acid positions (9, 63, and 233). These results imply that the ApxSC genes show co-dominant expression resulting from multiple alleles. This hypothesis is supported by genomic blots and primer extension analyses.

Characterization of trichobakin, a type I ribosome-inactivating protein from Trichosanthes sp. Bac Kan 8-98.

We have isolated a genomic clone encoding trichobakin (TBK), a type I ribosome-inactivating protein from the plant Trichosanthes sp. Bac Kan 8-98 (family Cucurbitaceae), by PCR using specific primers designed from the cDNA sequences of alpha-trichosanthin. The sequence encoding mature TBK was constructed in the pET-21d(+) vector for overexpression in Escherichia coli strain BL21(DE3). The recombinant protein was purified to homogeneity by CM-Sepharose chromatography on FPLC with a final yield of about 55 mg/l of culture. The protein has a molecular mass of about 27 kDa, as shown by SDS/PAGE and matrix-assisted laser-desorption ionization MS. It was found that the protein inhibited luciferase mRNA translation in the rabbit reticulocyte cell-free system with an IC(50) value (that which causes a 50% reduction of residual translational activity) of about 3.5 pM. The rRNA N-glycosidase activity of the protein was also proved at the above-mentioned concentration after rRNAs were treated with acid aniline.

Novel anther-specific myb genes from tobacco as putative regulators of phenylalanine ammonia-lyase expression.

Two cDNA clones (NtmybAS1 and NtmybAS2) encoding MYB-related proteins with strong sequence similarity to petunia (Petunia hybrida) PhMYB3 were isolated from a tobacco (Nicotiana tabacum cv Samsun) pollen cDNA library. Northern blot and in situ hybridization revealed that NtmybAS transcripts are specifically expressed in both sporophytic and gametophytic tissues of the anther including tapetum, stomium, vascular tissue, and developing pollen. Random binding site selection assays revealed that NtMYBAS1 bound to DNA sequences closely resembling consensus MYB binding sites MBSI and MBSIIG, with a higher affinity for MBSI. Transient expression analyses of the N-terminal MYB domain demonstrated the presence of functional nuclear localization signals, and full-length NtMYBAS1 was able to activate two different phenylalanine ammonia-lyase promoters (PALA and gPAL1) in tobacco leaf protoplasts. Similar analysis of truncated NtmybAS1 cDNAs identified an essential, C-terminal trans-activation domain. Further in situ hybridization analyses demonstrated strict co-expression of NtmybAS and gPAL1 in the tapetum and stomium. Despite abundant expression of NtmybAS transcripts in mature pollen, gPAL1 transcripts were not detectable in pollen. Our data demonstrate that NtMYBAS1 is a functional anther-specific transcription factor, which is likely to be a positive regulator of gPAL1 expression and phenylpropanoid synthesis in sporophytic, but not in gametophytic, tissues of the anther.

Molecular characterization of cDNA clones for ADP-glucose pyrophosphorylase from Citrus.

Two cDNA clones encoding ADP-glucose pyrophosphorylases have been isolated from fruit and leaf cDNA libraries of Citrus (Citrus unshiu Mac. cv. Miyagawa) in which one was designated as agpS for the small subunit and the other as agpL for the large subunit. Both cDNAs have uninterrupted open reading frames deriving 57-58 kDa polypeptides. The deduced amino acid sequence of agpS has a unique feature. That is, it lacks a cysteine residue (Cys-12) which is usually conserved in all other dicot enzymes. This is the first report of agpS lacking Cys-12 among dicot small subunits. The expression pattern of both subunits showed a different profile in which leaves synthesized both agpS and agpL more vigorously than those of fruits. During leaf development, the transcripts of agpS and agpL showed a higher expression level at younger stages. During fruit development, the expression level of both subunits was observed to be highest in the mini-green stage, but it decreased in the small green stage and it increased again towards the maturing stage. These results suggest that both subunits may play an important role in the regulation of Citrus fruit and leaf development.

Isolation of a cDNA encoding a 31-kDa, pathogenesis-related 5/thaumatin-like (PR5/TL) protein abundantly expressed in apple fruit (Nalus domestica cv. Fuji).

A fruit-specific and pathogenesis-related 5/thaumatin-like (PR5/TL), 31-kDa protein was isolated by 2D-PAGE from fully-grown apples (Malus domestica cv. Fuji) and named Mdtl1 (Malus domestica thaumatin-like protein 1). Using the N-terminal sequence of the protein, the full-length cDNA encoding Mdtll was isolated. The cDNA clone (Mdtl1) consists of 944 bp with an open reading frame (ORF) of 744 bp encoding a protein of 247 amino acids. The deduced amino acid sequence of Mdtl1 shows high similarity to the sequences of PR5/TL proteins. Mdtl1 is a slightly acidic protein with a putative signal peptide and a putative N-glycosylation site, and lacks a C-terminal extension. This suggests that Mdtl1 is an apoplastic glycoprotein. Results of northern blotting indicated that expressions of Mdtl1 are developmentally regulated. Southern blot analysis showed that Mdtl1 may be present as a single copy, and there exist other genes closely related to Mdtl1 in the apple genome.

Expression of the chloroplast-localized small heat shock protein by oxidative stress in rice.

A rice (Oryza sativa L. cv. Nakdong) cDNA clone, Oshsp26, encoding the chloroplast-localized small heat shock protein (smHSP) was isolated. Southern blot analysis of genomic DNA and the result of screening of a cDNA library indicated that the Oshsp26 gene is encoded by a single gene in the rice genome. The Oshsp26 gene was expressed following heat stress: the transcript level was highest when rice leaves were treated at high temperatures for 2h at 42 degrees C, and the transcripts became detectable after 20min and reached a maximum level after 2h. It was also found that the Oshsp26 gene was expressed following oxidative stress even in the absence of heat stress. Treatment of rice plants with methyl viologen (MV) in the light and treatment with hydrogen peroxide (H(2)O(2)), either in the light or in the dark, both caused a significant accumulation of the transcripts and the protein. Since MV treatment in the light leads to the generation of H(2)O(2) inside the chloroplast, it is likely that H(2)O(2) by itself acts to induce the expression of the Oshsp26 gene. These results suggest that the chloroplast smHSP plays an important role in protecting the chloroplast against damage caused by oxidative stress as well as by heat stress.

Differential roles of the 5' untranslated regions of cucumber mosaic virus RNAs 1, 2, 3 and 4 in translational competition.

RNA species of plant tripartite RNA viruses show distinct translational activities in vitro when the viral RNA concentration is high. However, it is not known what causes the differential translation of virion RNAs. Using an in vitro wheat germ translation system, we investigated the translation efficiencies and competitive activities of chimeric cucumber mosaic virus (CMV) RNAs that contained viral untranslated regions (UTRs) and a luciferase-coding sequence. The chimeric RNAs exhibited distinct translation efficiencies and competitive activities. For example, the translation of chimeric CMV RNA 4 was about 40-fold higher than that of chimeric CMV RNA 3 in a competitive environment. The distinct translation resulted mainly from differences in competitive activities rather than translation efficiencies of the chimeric RNAs. The differential competitive activities were specified by viral 5 UTRs, but not by 3 UTRs or viral proteins. The competitive translational activities of the 5 UTRs were as follows: RNA 4 (coat protein)>RNAs 2 and 1 (2a and 1a protein, or replicase )> RNA 3 (3a protein).

Functional MR imaging of working memory in the human brain.

OBJECTIVE: In order to investigate the functional brain anatomy associated with verbal and visual working memory, functional magnetic resonance imaging was performed. MATERIALS AND METHODS: In ten normal right handed subjects, functional MR images were obtained using a 1.5-T MR scanner and the EPI BOLD technique. An item recognition task was used for stimulation, and during the activation period of the verbal working memory task, consonant letters were used. During the activation period of the visual working memory task, symbols or diagrams were employed instead of letters. For the post-processing of images, the SPM program was used, with the threshold of significance set at p <.001. We assessed activated brain areas during the two stimulation tasks and compared the activated regions between the two tasks. RESULTS: The prefrontal cortex and secondary visual cortex were activated bilaterally by both verbal and visual working memory tasks, and the patterns of activated signals were similar in both tasks. The superior parietal cortex was also activated by both tasks, with lateralization to the left in the verbal task, and bilaterally without lateralization in the visual task. The inferior frontal cortex, inferior parietal cortex and temporal gyrus were activated exclusively by the verbal working memory task, predominantly in the left hemisphere. CONCLUSION: The prefrontal cortex is activated by two stimulation tasks, and this is related to the function of the central executive. The language areas activated by the verbal working memory task may be a function of the phonological loop. Bilateral prefrontal and superior parietal cortices activated by the visual working memory task may be related to the visual maintenance of objects, representing visual working memory.

Characterization of the full-length sequences of phospholipase A2 induced during flower development.

The suppression subtractive hybridization (SSH) method was used to isolate developmentally regulated genes during carnation flower maturation. Carnation flower maturation-related clones obtained by the SSH were serially assigned as CFMI (carnation flower maturation-induced) clones. Northern blot analysis showed that several CFMI clones were differentially expressed during flower development. One of the clones, CFMI-3, showed similarity to various animal secretory phospholipases A2 (PLA2). Since little is known about PLA2 gene sequence in plant species, the CFMI-3 clone was selected for further characterization by sequence analysis. Full sequence analysis reveals that the CFMI-3 contains a Ca2+ binding domain, a PLA2 active site, and 12 conserved Cys residues, which is a distinct characteristic of PLA2. Amino acid sequence alignment of CFMI-3 to various putative plant PLA2 confirmed that the CFMI-3 cDNA is the full-length putative PLA2 cDNA identified in plant species.

WAPK, a Ser/Thr protein kinase gene of Nicotiana tabacum, is uniquely regulated by wounding, abscisic acid and methyl jasmonate.

A cDNA encoding a protein kinase, which may be involved in the wound signal transduction pathway, was isolated from Nicotiana tabacum. The cDNA, named WAPK, is 1227 bp in length and contains an ORF of 1017 bp. The ORF encodes a polypeptide of 339 amino acids, with a calculated molecular mass of 38234 Da. Analysis of the deduced amino acid sequence shows that the N-terminal region of WAPK contains a catalytic region composed of eleven subdomains which are typically found in Ser/Thr protein kinases. This region shows 78-84% sequence identity with similar regions of abscisic acid (ABA)-induced and external-stimuli-responsive protein kinases. However, the C-terminal region of WAPK shows little homology with similar regions of Ser/Thr protein kinases, except for a 16-amino acid stretch near the end of the catalytic domain. Kinase assays using a WAPK fusion protein expressed in E. coli revealed that WAPK autophosphorylates on serine residue(s). The WAPK gene is predominantly expressed in flowers, moderately in roots, and poorly in leaves. Transcripts were not detected in stems. The WAPK gene was induced by wounding (within 1.5 h), by abscisic acid (within 0.5 h), and by methyl jasmonate (within 2 h). The induction pattern of WAPK mRNA upon wounding was not affected by treatment with diethyldithiocarbamic acid, a reagent which inhibits jasmonic acid biosynthesis. These results suggest that the WAPK gene is regulated by ABA in the wound signal transduction pathway.

Isolation of genomic DNA containing a cytosolic ascorbate peroxidase gene (ApxSC) from the strawberry (Fragaria x ananassa).

We isolated a genomic DNA harboring a cytosolic ascorbate peroxidase gene (ApxSC) from a genomic library of the strawberry (Fragaria x ananassa). Restriction mapping and sequence analyses showed that the DNA is composed of 2.5 kb of the full-length ApxSC gene, 3.7 kb of the 5'-upstream region, and 0.5 kb of the 3'-downstream region. The ApxSC genomic DNA contains 10 exons and 9 introns, which is similar to the structure of pea ApxI. A primer extension analysis suggested that the transcription of ApxSC gene was started at three start sites with different degrees. The promoter region of ApxSC gene contains a sequence or structure distinct from other reported plant ascorbate peroxidase genes, though with several known functional elements such as a TATA box.

Characterization of cDNAs encoding small and large subunits of ADP-glucose pyrophosphorylases from watermelon (Citrullus vulgaris S.).

Three cDNA clones encoding ADP-glucose pyrophosphorylases were isolated from a full red fruit cDNA library of watermelon (Citrullus vulgaris S.). Sequence analyses indicated that one clone, wms1, corresponds to the small subunit, and two clones, wml1 and wml2 (a partial gene), are the large subunits of AGPase. The presumed AGPase proteins encoded by wms1, wml1, and wml2 have 526, 526, and 481 amino acids, respectively. The protein sequences have the conserved amino acids important for the substrate or regulator binding site, with some variation. Developmental changes in the amounts of wms1, wml1, and wml2 transcripts in fruits were measured by northern blot analysis. Their expression levels decreased from the small green to medium green stages, then increased in accordance with fruit ripening, which was different from those of tomato and oriental melon.

Molecular cloning and organ-specific expression of three isoforms of tomato ADP-glucose pyrophosphorylase gene.

We isolated three cDNAs encoding different isoforms of ADP-glucose pyrophosphorylase (AGP) large submits from tomato plants. Three clones, designated AgpL1, AgpL2, and AgpL3 were 2019, 2105, and 1850bp, respectively. The clones had a long, uninterrupted open reading frame with a start codon at the 5' region and different copies of polyadenylation signal (AATAAA) at the 3' region, deriving 57-58kDa polypeptides. Sequence comparison and phylogenetic analysis revealed that the three isoforms represented different types of AGP large subunits, AgpL1 was strongly expressed in stems and weakly in roots. Accumulation of AgpL1 transcripts was found even in unpollinated ovaries and sustained at the early stages of fruit development. ApgL2 was expressed in roots and fruits. AgpL3 was exclusively expressed in leaves. The present study suggests that the three isoforms of tomato AGP large subunits are organ-specific in their expressions.

The 5' untranslated region of cucumber mosaic virus RNA4 confers highly competitive activity on heterologous luciferase mRNA in cell-free translation.

The cell-free translation of virion RNAs of several tripartite RNA viruses has shown that RNA4, a subgenomic RNA, is more competitive than other virion RNAs. Recently, the 3' untranslated region (UTR) of alfalfa mosaic virus (AMV) RNA4 was identified to be a competitive determinant. In this study, we observed that the RNA4 of cucumber mosaic virus (CMV), another tripartite RNA virus, was also found to be a strong competitor in translational competition among CMV virion RNAs. To identify the competitive determinant of CMV RNA4, we constructed various chimeric luciferase mRNAs containing RNA4 and/or vector-derived UTRs. The relative translations of luciferase-containing mRNA in the presence of a competitor mRNA showed that the 5' UTR, not the 3' UTR, substantially contributed to the highly competitive activity of CMV RNA4.

Isolation of ALU1-P gene encoding a protein with aluminum tolerance activity from Arthrobacter viscosus.

We isolated a DNA fragment (ALU1-P) encoding a protein with an activity of aluminum tolerance from an Al tolerant soil microorganism, Arthrobacter viscosus. This microorganism was isolated from acidic tea field soils. The cloned DNA is composed of 1090 nucleotides, which has one open-reading frame without any stop codon. However, when the DNA fragment was transferred into Escherichia coli, a microorganism susceptible to Al toxicity, it endowed E. coli with Al tolerance. The deduced amino acid sequence of the DNA showed 65% identity with the protein of YbaX gene in Escherichia coli, and 51.1% identity with YB91 Haein hypothetical protein of HI1191 gene in Haemophilus influenzae. The ALU1-P gene in the expression vector produced a protein of 192 amino acids deriving a molecular weight of 21.3 kDa by using the stop codon in vector. The ALU1-P gene is a new one that has the characteristic of Al tolerance.

Ribozyme mediated targeting of cucumber mosaic virus RNA 1 and 2 in transgenic tobacco plants.

Hammerhead ribozymes have been extensively used to inhibit the expression of cellular genes or viral genes mainly in the animal study. In this study, we designed a ribozyme targeting the conserved leader sequences of cucumber mosaic virus (CMV) RNA 1 and 2. The ribozyme, with asymmetric lengths of flanking complementary regions, cleaved a model substrate RNA efficiently at 26 degrees C as well as at 37 degrees C or 50 degrees C in vitro. And the ribozyme encoding sequence was introduced into tobacco plants and expressed with the CaMV 35S promoter and 3' NOS terminator in a monomeric type (pBIR1), tandemly repeated type (pBIR3), and cotranscriptionally combined type (pRokR) with 2.2 copies of I17N satellite RNA. Virus challenging experiments in F1 plants of respective transformants with CMV-Y showed specific reductions of viral RNA 1 and 2 in comparison with RNA 3 or 4. Although young plants of a three-leaf-stage showed rather similar mild symptom attenuations in all constructions compared to CMV-Y inoculated wild type, fully grown plants showed a differential degree of resistance upon systemic infections of CMV-Y in pRokR, pBIR3 and pBIR1 transformed plants in a decreasing order.