Stable Plastid Transformation in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes—aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)—in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.     

Effect of seed-specific expression of the <Emphasis Type="Italic">ipt</Emphasis> Gene on <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. seed composition

A transgenic approach to manipulation of endosperm development has been investigated. Nicotiana tabacum cv. Xanthi, an endosperm-containing dicotyledon, has been used as a model plant and the 2.6 kb wheat high molecular weight (HMW) glutenin subunit 12 promoter has been used fused either to the gus reporter gene (HMWgus construct)—to study promoter characteristics—or to the Agrobacterium ipt gene—to study the effect of cytokinin (CK) over-expression on assimilate accumulation in the seed. In transgenic tobacco the promoter:gus fusion showed that HMW is an endosperm-specific promoter with maximum expression 20 days after anthesis (DAA), corresponding to the mid to late stages of seed development. Transgenic plants containing the HMWipt construct showed no morphological abnormalities but they had an average increase in seed weight and total ethanol-insoluble carbohydrates and protein content of 8.1%, 7.0% and 8.3%, respectively. SDS PAGE analysis demonstrated that the effect on protein accumulation was non-specific. The highest values of the parameters analysed correlated with moderate increases in the levels of biologically active CKs. These results suggest that ectopic expression of small amounts of CKs can be used to increase storage assimilate accumulation without a detrimental effect on development.     

Overexpression and Inactivation of <Emphasis Type="Italic">UGT73B2</Emphasis> Modulate Tolerance to Oxidative Stress in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Glycosyltransferases (GTs) play an important role in modulating solubility, stability, bioavailability, and bioactivity of secondary metabolites, such as flavonoids. In Arabidopsis thaliana, at least 120 family 1 uridine diphosphate (UDP)-glycosyltransferases (UGTs) have been predicted. However, little is known about their substrates or their physiological roles in planta. To define the role of UGT73B2 in planta, we first characterized its expression pattern using transgenic Arabidopsis plants carrying the cis-elements of UGT73B2 fused to the GUS reporter. During vegetative phase, its expression was high in embryonic and postembryonic roots, where it may play a physiological role in the glycosylation of flavonoids. Loss of function of UGT73B2 alone or in conjunction with its closest homologs, UGT73B1 and UGT73B3, confers greater tolerance to oxidative stress, whereas overexpression of UGT73B2 increases sensitivity to oxidative stress. In addition, growth phenotypes of mutant and transgenic seedlings correlate well with ROS levels in planta. Our results suggest that the glycosylation of flavonoids by UGT73B2—and/or its closest homologs—modulate the response of plants to oxidative stress.     

<Emphasis Type="Italic">Hox</Emphasis> cluster duplication in the basal teleost <Emphasis Type="Italic">Hiodon alosoides</Emphasis> (Osteoglossomorpha)

Large-scale—even genome-wide—duplications have repeatedly been invoked as an explanation for major radiations. Teleosts, the most species-rich vertebrate clade, underwent a “fish-specific genome duplication” (FSGD) that is shared by most ray-finned fish lineages. We investigate here the Hox complement of the goldeye (Hiodon alosoides), a representative of Osteoglossomorpha, the most basal teleostean clade. An extensive PCR survey reveals that goldeye has at least eight Hox clusters, indicating a duplicated genome compared to basal actinopterygians. The possession of duplicated Hox clusters is uncoupled to species richness. The Hox system of the goldeye is substantially different from that of other teleost lineages, having retained several duplicates of Hox genes for which crown teleosts have lost at least one copy. A detailed analysis of the PCR fragments as well as full length sequences of two HoxA13 paralogs, and HoxA10 and HoxC4 genes places the duplication event close in time to the divergence of Osteoglossomorpha and crown teleosts. The data are consistent with—but do not conclusively prove—that Osteoglossomorpha shares the FSGD. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

In vivo effects of <Emphasis Type="Italic">NbSiR</Emphasis> silencing on chloroplast development in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Sulfite reductase (SiR) performs dual functions, acting as a sulfur assimilation enzyme and as a chloroplast (cp-) nucleoid binding protein. In this study, we examined the in vivo effects of SiR deficiency on chloroplast development in Nicotiana benthamiana. Virus-induced gene silencing of NbSiR resulted in leaf yellowing and growth retardation phenotypes, which were not rescued by cysteine supplementation. NbSiR:GFP fusion protein was targeted to chloroplasts and colocalized with cp-nucleoids. Recombinant full-length NbSiR protein and the C-terminal half of NbSiR possessed cp-DNA compaction activities in vitro, and expression of full-length NbSiR in E. coli caused condensation of genomic DNA. NbSiR silencing differentially affected expression of plastid-encoded genes, inhibiting expression of several genes more severely than others. In the later stages, depletion of NbSiR resulted in chloroplast ablation. In NbSiR-silenced plants, enlarged cp-nucleoids containing an increased amount of cp-DNA were observed in the middle of the abnormal chloroplasts, and the cp-DNAs were predominantly of subgenomic sizes based on pulse field gel electrophoresis. The abnormal chloroplasts developed prolamellar body-like cubic lipid structures in the light without accumulating NADPH:protochlorophyllide oxidoreductase proteins. Our results suggest that NbSiR plays a role in cp-nucleoid metabolism, plastid gene expression, and thylakoid membrane development.     

Molecular cloning and daily variations of the <Emphasis Type="Italic">Period</Emphasis> gene in a reef fish <Emphasis Type="Italic">Siganus guttatus</Emphasis>

As the first step in understanding the molecular oscillation of the circa rhythms in the golden rabbitfish Siganus guttatus—a reef fish with a definite lunar-related rhythmicity—we cloned and sequenced a Period gene (rfPer). The rfPer gene contained an open reading frame that encodes a protein consisting of 1,452 amino acids; this protein is highly homologous to PER proteins of vertebrates including zebrafish. Phylogenetic analyses indicated that the rfPER protein is related to the zebrafish PER1 and PER4. The expression of rfPer mRNA in the whole brain, retina, and liver under light/dark (LD) conditions increased at 06:00 h and decreased at 18:00 h, suggesting that its robust circadian rhythm occurs in neural and peripheral tissues. When daily variation in the expression in rfPer mRNA in the whole brain and cultured pineal gland were examined under LD conditions, similar expression patterns of the gene were observed with an increase around dawn. Under constant light condition, the increased expression of rfPer mRNA in the whole brain disappeared around dawn. The present results demonstrate that rfPer is related to zPer4 and possibly zPer1. The present study is the first report on the Period gene from a marine fish.     

Isolation and <Emphasis Type="Italic">S</Emphasis>-genotyping application of <Emphasis Type="Italic">S</Emphasis>-allelic polymorphic <Emphasis Type="Italic">MdSLFB</Emphasis>s in apple (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.)

Apple (Malus domestica Borkh.) possesses gametophytic self-incompatibility (GSI) which is controlled by S-RNase in the pistil as well as a pollen S-determinant that has not been well characterized. The identification of S-locus F-box brother (SFBB) genes, which are good candidates for the pollen S-determinant in apple and pear, indicated the presence of multiple S-allelic polymorphic F-box genes at the S-locus. In apple, two SFBB gene groups have been described, while there are at least three groups in pear. In this report, we identified five MdSLFB (S-RNase-linked F-box) genes from four different S-genotypes of apple. These genes showed pollen- and S-allele-specific expression with a high polymorphism among S-alleles. The phylogenetic tree suggested that some of them belong to SFBBα or β groups as described previously, while others appear to be different from SFBBs. In particular, the presence of MdSLFB3 and MdSLFB9 suggested that there are more S-allelic polymorphic F-box gene groups in the S-locus besides α and β. Based on the sequence polymorphism of MdSLFBs, we developed an S-genotyping system for apple cultivars. In addition, we isolated twelve MdSLFB-like genes, which showed pollen-specific expression without S-allelic polymorphism.     

Expression profiles of glutathione <Emphasis Type="Italic">S</Emphasis>-transferase genes in larval midgut of <Emphasis Type="Italic">Bombyx mori</Emphasis> exposed to insect hormones

Glutathione S-transferases (GSTs) are believed to play a role in the detoxification of xenobiotics, resistance to insect viruses and pesticides, intracellular transport, biosynthesis of hormones and protection against oxidative stress. In this study, we used quantitative real time RT-PCR to examine expression profiles of the silkworm Bombyx mori GST-Sigma (BmGSTS2) and GST-Delta (BmGSTD2) genes in the larval midgut of the silkworm after exposure to 2-hydroxyecdysone (20E) and juvenile hormone analog (JHA). In concentration-course study, 20E at higher concentrations (1.0 and 2.0 μg/μl) caused significant upregulation of BmGSTD2, and all concentrations (0.5–2.0 μg/μl) of 20E caused significant upregulation of BmGSTS2. However, JHA in all concentrations downregulated the expression of BmGSTD2 and BmGSTS2. When exposed to either 20E (2.0 μg/μl) or JHA (2.0 μg/μl) on the third day of the fifth instar, the silkworm had higher BmGSTD2 at later time points: 15, 18, and 24 h for 20E and 24 h for JHA. BmGSTS2 expression was downregulated within 24 h after exposure to JHA and showed a time-dependent response after exposure to 20E. We also did a stage-dependent study, in which JHA downregulated BmGSTD2 expression and upregulated BmGSTS2 expression significantly at both day 1 and day 3 of the fifth instar. 20E upregulated the expression of BmGSTD2 and BmGSTS2 at the two stages. These findings imply that hormones have an important role in the regulation of basal GST expression. However, further validation and field trials should be carried out on the regulatory elements relevant to BmGSTD2 and BmGSTS2 gene expression.     

Pollen-Specific Expression of <Emphasis Type="Italic">Oryza sativa Indica</Emphasis> Pollen Allergen Gene (<Emphasis Type="Italic">OSIPA</Emphasis>) Promoter in Rice and <Emphasis Type="Italic">Arabidopsis</Emphasis> Transgenic Systems

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.     

Stable chloroplast transformation in cabbage (<Emphasis Type="Italic">Brassica oleracea </Emphasis>L. var. <Emphasis Type="Italic">capitata </Emphasis>L.) by particle bombardment

The objectives of this research were first to isolate plastid gene sequences from cabbage (Brassica oleracea L. var. capitata L.), and to establish the chloroplast transformation technology of Brassica. A universal transformation vector (pASCC201) for Brassica chloroplast was constructed with trnV–rrn16S (left) and trnI–trnA–rrn23S (right) of the IR_A region as a recombination site for the transformed gene. In transforming plasmid pASCC201, a chimeric aadA gene was cloned between the rrn16S and rrn23S plastid gene borders. Expression of aadA confers resistance to spectinomycin and streptomycin antibiotics. The uidA gene was also inserted into the pASCC201 and transferred into the leaf cells of cabbage via particle gun mediated transformation. Regenerated plantlets were selected by 200 mg/l spectinomycin and streptomycin. After antibiotic selection, the regeneration percentage of the two cabbage cultivars was about 2.7–3.3%. The results of PCR testing and Southern blot analysis confirmed that the uidA and aadA genes were present in the chloroplast genome via homologously recombined. Northern blot hybridizations, immunoblotting and GUS histochemical assays indicated that the uidA gene were stable integrated into the chloroplast genome. Foreign protein was accumulated at 3.2–5.2% of the total soluble protein in transgenic mature leaves. These results suggest that the expression of a variety of foreign genes in the chloroplast genome will be a powerful tool for use in future studies.     

<Emphasis Type="Italic">Elaeocarpus</Emphasis> for <Emphasis Type="Italic">Flora Malesiana</Emphasis>: new taxa and understanding in the <Emphasis Type="Italic">Ganitrus</Emphasis> group

The Ganitrus group of Elaeocarpus L. (incl. Elaeocarpus Sect. Ptilanthus Schltr.) as found in Malesia is examined and some understanding reached. The group is more complex and speciose in eastern Malesia, especially in Papuasia (with the Philippines, Sulawesi and Maluku somewhat less rich) than in western and central Malesia. E. angustifolius Blume (syn. E. ganitrus & E. sphaericus auctt. mult.) — the source of the ornamental beads known as rudraksha of eastern commerce, from which malas or rosaries are made — is considered to be very variable in Malesia and probably varies further in the extremes of its range; outside Malesia the Ganitrus group is represented by ‘E. angustifolius s.l.’ only. The following species are recognised: E. altisectus Schltr. with a new subsp. carrii Coode from New Guinea; E. angustifolius Blume; E. avium Coode sp. nov. from New Guinea; E. buderi Coode; E. dolichostylus Schltr. with E. chloranthus treated as var. chloranthus (A. C. Sm.) Coode from New Guinea, with a new var. elliptifolius Coode from the Talaud/Sangihe Is. and a new var. hentyi Coode (replacing and re-interpreting subsp. collinus Coode) from New Guinea; E. fulvus Elmer; E. kaniensis Schltr.; E. ornatus Coode sp. nov. from New Guinea; E. osiae Coode sp. nov. from Bougainville I.; E. ptilanthus Schltr.; E. ramiflorus Merr. and E. trichopetalus Merr. & Quisumb. — a total of 12 species although the last two are perhaps not truly distinct from E. angustifolius. The stone sculpturing is discussed and some useful characters revealed. Attention is drawn to a collection from mid-altitude in Papua New Guinea, inadequately known but probably a new species in this group.     

Chloroplast-located <Emphasis Type="Italic">BjFer1</Emphasis> together with anti-oxidative genes alleviate hydrogen peroxide and hydroxyl radical injury in cytoplasmic male-sterile <Emphasis Type="Italic">Brassica juncea</Emphasis>

We studied how plant cell modulated redox homeostasis in cytoplasmic male-sterility (CMS) Brassica juncea. The CMS Brassica juncea was identified to be mutated in several mitochondrial genes that suggested the changes of cell redox homeostasis. We observed that it was not associated with increased oxidative stress as shown by decreased H₂O₂ and ^∙OH contents in this type of CMS. The expressions of several anti-oxidative genes were up-regulated in 5-day-old seedlings of CMS than MF lines under light and dark conditions. The mitochondrial alternative oxidase pathway was not activated, as indicated by no increased expression of AOX1a gene in CMS. Interestingly, the expression of Ferritin1 gene was markedly activated in 5-day-old seedlings of CMS than MF line under light and dark conditions. Consequently, we detected increased content of total iron in 30-day-old leaves in CMS than MF line. We isolated Ferritin1 orthologous gene from Brassica juncea, which was targeted to the chloroplast and induced by Fe-citrate and H₂O₂, not ABA. Taken together, we proposed that increased expressions of BjFer1 and several antioxidant genes protected cell from oxidative stress in CMS Brassica juncea.