Molecular cloning and characterization of five genes encoding pentatricopeptide repeat proteins from Upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

The pentatricopeptide repeat (PPR) protein family is one of the largest and most complex families in plants. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding RNA. PPR proteins have been implicated in many crucial functions broadly involving organelle biogenesis and plant development. In this study, we identified many genes encoding PPR protein in Upland cotton through an extensive survey of the database of Gossypium hirsutum. Furthermore, we isolated five full-length cDNA of PPR genes from G. hirsutum 0-613-2R which were named GhPPR1–GhPPR5. Domain analysis revealed that the deduced amino acid sequences of GhPPR1–5 contained from 5 to 10 PPR motifs and those PPR proteins were divided into two different PPR subfamilies. GhPPR1–2 belonged to the PLS subfamily and GhPPR3–5 belonged to the P subfamily. Phylogenetic analysis of the five GhPPR proteins and 18 other plant PPR proteins also revealed that the same subfamily clustered together. All five GhPPR genes were differentially but constitutively expressed in roots, stems, leaves, pollens, and fibers based on the gene expression analysis by real-time quantitative RT-PCR. This study is the first report and analysis of genes encoding PPR proteins in cotton.     

Induction of pathogenesis-related proteins during biocontrol of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> with <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.) plants

To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation, when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa. Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms.     

High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and β-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54–95% of the cotyledon explants on MXB selective medium containing 200 μg ml⁻¹ kanamycin and 500 μg ml⁻¹ carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4–5 weeks after inoculation. Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode. Received: 13 July 1999 / Accepted: 8 August 1999     

Developmental and environmental regulation of soybean <Emphasis Type="Italic">SE60</Emphasis> gene expression during embryogenesis and germination

Soybean SE60 belongs to the γ-thionin family of proteins. We recently demonstrated that SE60 plays a role in defense during soybean development. Here, we show that SE60 is expressed in a tissue-specific and developmentally regulated manner. The expression of SE60 is distinct from that of the glycinin (Gy2) and extensin (SbHRGP3) genes of soybean during embryogenesis and germination. A SE60::GUS(−809) transgene, comprising −809 bp of the 5′-flanking region of SE60 fused to the GUS reporter gene, was expressed specifically in developing embryos, but not in the endosperms, from the globular stage of transgenic tobacco and Arabidopsis seeds. Furthermore, light affected the SE60::GUS(−809) expression pattern in germinating seedlings. Electrophoretic mobility shift assay (EMSA) revealed that soybean nuclear proteins as well as E. coli-expressed SB16, a high mobility group protein (HMG), were bound sequence-specifically to the fragment containing AT-rich motifs identified in the SE60 promoter. Interestingly, the soybean nuclear proteins binding to the two G-boxes and RY repeat were prevalent in seeds of 2–4 mm in size. In contrast, the nuclear proteins binding to the AT-rich motif and SE60 RNA expression were more prominent in seeds of 4–6 mm in size. Therefore, we propose that factors binding to the G-boxes or RY repeat initiate SE60 expression during embryogenesis.     

Genetic variation of the white-spotted longicorn beetle <Emphasis Type="Italic">Anoplophora</Emphasis> spp. (Coleoptera: Cerambycidae) in Japan detected by mitochondrial DNA sequence

Genetic variation of the white-spotted longicorn beetle Anoplophora spp., distributed on the Japanese mainland and the Ryukyu Islands, was examined using nucleotide sequences of mitochondrial DNA. Two fragments, a 1.2-kb-long fragment containing portions of the cytochrome oxidase subunit I and II genes and a 1.4-kb-long fragment containing portions of 16S and 12S rDNAs, were sequenced. In phylogenetic analyses based on the sequences, 294 individuals collected at 75 localities were divided into two major groups (groups A and B) and then split into seven subgroups (A1–A4 and B1–B3). In group A, the closely related subgroups A1, A2, and A3 were mainly distributed on the Japanese mainland and were roughly separated among geographic areas, although the range of A3 spread significantly from Kyushu to the Central Ryukyu Islands. The sequence of A4, detected from one individual collected in eastern Honshu, was almost the same as that reported for A. chinensis (Forster) on the Chinese continent. In group B, subgroups B1 and B2 were restricted to the Central and Southern Ryukyu Islands, respectively, while B3 was distributed widely in both regions. Based on the results, we discuss the geographic origin of the haplotypes and movement of the insects between geographic areas.     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.     

Ras-like Small GTPases Form a Large Family of Proteins in the Marine Sponge <Emphasis Type="Italic">Suberites domuncula</Emphasis>

Sponges (Porifera) are the simplest and the most ancient metazoan animals, which branched off first from the common ancestor of all multicellular animals. We have inspected ∼13,000 partial cDNA sequences (ESTs) from the marine sponge Suberites domuncula and have identified full or partial cDNA sequences coding for ∼50 different Ras-like small GTPases. Forty-four sponge proteins from the Ras family are described here: 6 proteins from the Ras subfamily, 5 from Rho, 6 from Arf, 1 Ran, and 26 Rabs or Rab-like proteins. No isoforms of these proteins were detected; the closest related proteins are two Rho proteins with 74% identity. Small GTPases from sponge display a higher degree of sequence conservation with orthologues from vertebrates (53%–93% identity) than with those from either Caenorhabditis elegans or Drosophila melanogaster. The real number of small GTPases in this sponge is certainly much higher than 50, because the actual S. domuncula database of ∼13,000 ESTs contains at most 3000 nonredundant cDNA sequences. The number of genes for Ras-like small GTPases in yeast, C. elegans, D. melanogaster, and humans is 30, 56, 90, and 174, respectively. Both model invertebrates have only 29 Rabs or Rab-like proteins, compared with 26 already found in sponge, and are missing at least 1 Rab (Rab24) present in S. domuncula and mammals. Our results indicate that duplications and diversifications of genes encoding Ras-like small GTPases, especially the Rab subfamily of small GTPases, happened very early in the evolution of Metazoa. Reviewing Editor: Dr. Martin Kreitman Vera Gamulin passed away on 12 October 2006. We feel privileged to have had the opportunity to have worked with her.     

Expression of Two Old Yellow Enzyme Homologues from <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> and Identification of their Citral Hydrogenation Abilities

Two genes that encode proteins which share 30–35% sequence identity with yeast OYE (Old Yellow Enzyme, an NAD(P)H FMN-oxidoreductase), the well-studied archetype of the OYE protein family, have been identified in Gluconobacter oxydans M5. The two genes are localized in the chromosome and plasmid, respectively. Comparison of the deduced amino acid sequences of the enzymes with database entries revealed 75.1% similarity and 64.9% identity to that of the Pseudomonas syringae pv. glycinea NAD(P)H-dependent 2-cyclohexen-1-one reductase. The two proteins were expressed as His-tag fusion proteins in Escherichia coli and purified. The ability of the purified proteins to hydrogenate citral was identified. The results showed that the α,β-double bond of citral cis-isomer ‘neral’ could be stereoselectively reduced to produce citronellal by the purified OYE homologues.     

Molecular Cloning and Characterization of Rubber Biosynthetic Genes from <Emphasis Type="Italic">Taraxacum koksaghyz</Emphasis>

Rubber biosynthesis requires the action of specific enzymes known as cis-prenyltransferases (CPTs). These enzymes are responsible for the sequential addition of isopentenyl pyrophosphate units to the growing polyisoprene chain, a biochemical reaction thought to be stimulated by the presence of small rubber particle proteins (SRPPs). We have cloned, characterized, and analyzed the expression of three CPT genes (TkCPT1–3) and five SRPP genes (TkSRPP1–5) from the rubber-producing plant Taraxacum koksaghyz. The deduced TkCPT amino acid sequences showed significant levels of sequence identity with Hevea brasiliensis CPTs. We also found no obvious differences between SRPPs from T. koksaghyz, another rubber producer, and a non-rubber plant. The roles of the individual TkCPTs and TkSRRPs in rubber biosynthesis are discussed.     

Molecular and functional analyses of rice NHX-type Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter genes

We previously cloned a vacuolar Na⁺/H⁺ antiporter gene (OsNHX1) from rice (Oryza sativa). Here we identified four additional NHX-type antiporter genes in rice (OsNHX2 through OsNHX5) and performed molecular and functional analyses of those genes. The exon–intron structure of the OsNHX genes and the phylogenetic tree of the OsNHX proteins suggest that the OsNHX proteins are categorized into two subgroups (OsNHX1 through OsNHX4 and OsNHX5). OsNHX1, OsNHX2, OsNHX3, and OsNHX5 can suppress the Na⁺, Li⁺, and hygromycin sensitivity of yeast nhx1 mutants and their sensitivity to a high K⁺ concentration. The expression of OsNHX1, OsNHX2, OsNHX3, and OsNHX5 is regulated differently in rice tissues and is increased by salt stress, hyperosmotic stress, and ABA. When we studied the expression of β-glucuronidase (GUS) driven by either the OsNHX1 or the OsNHX5 promoter, we observed activity in the stele, the emerging part of lateral roots, the vascular bundle, the water pore, and the basal part of seedling shoots with both promoters. In addition, each promoter had a unique expression pattern. OsNHX1 promoter–GUS activity only was localized to the guard cells and trichome, whereas OsNHX5 promoter–GUS activity only was localized to the root tip and pollen grains. Our results suggest that the members of this gene family play important roles in the compartmentalization into vacuoles of the Na⁺ and K⁺ that accumulate in the cytoplasm and that the differential regulation of antiporter gene expression in different rice tissues may be an important factor determining salt tolerance in rice.     

Archaeal Communities in Boreal Forest Tree Rhizospheres Respond to Changing Soil Temperatures

Temperature has generally great effects on both the activity and composition of microbial communities in different soils. We tested the impact of soil temperature and three different boreal forest tree species on the archaeal populations in the bulk soil, rhizosphere, and mycorrhizosphere. Scots pine, silver birch, and Norway spruce seedlings were grown in forest humus microcosms at three different temperatures, 7–11.5°C (night–day temperature), 12–16°C, and 16–22°C, of which 12–16°C represents the typical mid-summer soil temperature in Finnish forests. RNA and DNA were extracted from indigenous ectomycorrhiza, non-mycorrhizal long roots, and boreal forest humus and tested for the presence of archaea by nested PCR of the archaeal 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) profiling and sequencing. Methanogenic Euryarchaeota belonging to Methanolobus sp. and Methanosaeta sp. were detected on the roots and mycorrhiza. The most commonly detected archaeal 16S rRNA gene sequences belonged to group I.1c Crenarchaeota, which are typically found in boreal and alpine forest soils. Interestingly, also one sequence belonging to group I.1b Crenarchaeota was detected from Scots pine mycorrhiza although sequences of this group are usually found in agricultural and forest soils in temperate areas. Tree- and temperature-related shifts in the archaeal population structure were observed. A clear decrease in crenarchaeotal DGGE band number was seen with increasing temperature, and correspondingly, the number of euryarchaeotal DGGE bands, mostly methanogens, increased. The greatest diversity of archaeal DGGE bands was detected in Scots pine roots and mycorrhizas. No archaea were detected from humus samples from microcosms without tree seedling, indicating that the archaea found in the mycorrhizosphere and root systems were dependent on the plant host. The detection of archaeal 16S rRNA gene sequences from both RNA and DNA extractions show that the archaeal populations were living and that they may have significant contribution to the methane cycle in boreal forest soil, especially when soil temperatures rise.     

Isolation and characterization of some novel genes of the apolipoprotein A-I family in Japanese eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>

Apolipoproteins such as apolipoprotein (apo) A-I, apoA-IV, and apoE are lipid binding proteins synthesized mainly in the liver and the intestine and play an important role in the transfer of exogenous or endogenous lipids through the circulatory system. To investigate the mechanism of lipid transport in fish, we have isolated some novel genes of the apoA-I family, apoIA-I (apoA-I isoform) 1–11, from Japanese eel by PCR amplification. Some of the isolated genes of apoIA-I corresponded to 28kDa-1 cDNAs which had already been deposited into the database and encoded an apolipoprotein with molecular weight of 28 kDa in the LDL, whereas others seemed to be novel genes. The structural organization of all apoIA-Is consisted of four exons separated by three introns. ApoIA-I10 had a total length of 3232 bp, whereas other genes except for apoIA-I9 ranged from 1280 to 1441 bp. The sequences of apoIA-Is at the exon-intron junctions were mostly consistent with the consensus sequence (GT/AG) at exon-intron boundaries, whereas the sequences of 3′ splice acceptor in intron 1 of apoIA-I1-7 were (AC) but not (AG). The deduced amino acid sequences of all apoIA-Is contained a putative signal peptide and a propeptide of 17 and 5 amino acid residues, respectively. The mature proteins of apoIA-I1-3, 7, and 8 consisted of 237 amino acids, whereas those of apoIA-I4-6 consisted of 239 amino acids. The mature apoIA-I10 sequence showed 65% identity to amino acid sequence of apoIA-I11 which was associated with an apolipoprotein with molecular weight of 23 kDa in the VLDL. All these mature apoIA-I sequences satisfied the common structural features depicted for the exchangeable apolipoproteins such as apoA-I, apoA-IV, and apoE but apoIA-I11 lacked internal repeats 7, 8, and 9 when compared with other members of apoA-I family. Phylogenetic analysis showed that these novel apoIA-Is isolated from Japanese eel were much closer to apoA-I than apoA-IV and apoE, suggesting new members of the apoA-I family.