A new galling insect model enhances photosynthetic activity in an obligate holoparasitic plant.

Insect-induced galls are microhabitats distinct from the outer environment that support inhabitants by providing improved nutrients, defence against enemies, and other unique features. It is intriguing as to how insects reprogram and modify plant morphogenesis. Because most of the gall systems are formed on trees, it is difficult to maintain them in laboratories and to comprehend the mechanisms operative in them through experimental manipulations. Herein, we propose a new model insect, Smicronyx madaranus, for studying the mechanisms of gall formation. This weevil forms spherical galls on the shoots of Cuscuta campestris, an obligate parasitic plant. We established a stable system for breeding and maintaining this ecologically intriguing insect in the laboratory, and succeeded in detailed analyses of the gall-forming behaviour, gall formation process, and histochemical and physiological features. Parasitic C. campestris depends on host plants for its nutrients, and usually shows low chlorophyll content and photosynthetic activity. We demonstrate that S. madaranus-induced galls have significantly increased CO2 absorbance. Moreover, chloroplasts and starch accumulated in gall tissues at locations inhabited by the weevil larvae. These results suggest that the gall-inducing weevils enhance the photosynthetic activity in C. campestris, and modify the plant tissue to a nutrient-rich shelter for them.

Author Correction: Oxidative rearrangement of (+)-sesamin by CYP92B14 co-generates twin dietary lignans in sesame.

The original version of of the Supplementary Information associated with this Article inadvertently omitted oligonucleotide primer sequences from Supplementary Table 3 and Supplementary Methods describing the molecular cloning of CYP92B14, CPR1 and CYP81Q cDNA fragments. The HTML has been updated to include a corrected version of the Supplementary Information.

Oxidative rearrangement of (+)-sesamin by CYP92B14 co-generates twin dietary lignans in sesame.

(+)-Sesamin, (+)-sesamolin, and (+)-sesaminol glucosides are phenylpropanoid-derived specialized metabolites called lignans, and are rich in sesame (Sesamum indicum) seed. Despite their renowned anti-oxidative and health-promoting properties, the biosynthesis of (+)-sesamolin and (+)-sesaminol remained largely elusive. Here we show that (+)-sesamolin deficiency in sesame is genetically associated with the deletion of four C-terminal amino acids (Del4C) in a P450 enzyme CYP92B14 that constitutes a novel clade separate from sesamin synthase CYP81Q1. Recombinant CYP92B14 converts (+)-sesamin to (+)-sesamolin and, unexpectedly, (+)-sesaminol through an oxygenation scheme designated as oxidative rearrangement of α-oxy-substituted aryl groups (ORA). Intriguingly, CYP92B14 also generates (+)-sesaminol through direct oxygenation of the aromatic ring. The activity of CYP92B14 is enhanced when co-expressed with CYP81Q1, implying functional coordination of CYP81Q1 with CYP92B14. The discovery of CYP92B14 not only uncovers the last steps in sesame lignan biosynthesis but highlights the remarkable catalytic plasticity of P450s that contributes to metabolic diversity in nature.

Involvement of auxin dynamics in hypergravity-induced promotion of lignin-related gene expression in Arabidopsis inflorescence stems.

Recent studies have shown that hypergravity enhances lignification through up-regulation of the expression of lignin biosynthesis-related genes, although its hormonal signalling mechanism is unknown. The effects of hypergravity on auxin dynamics were examined using Arabidopsis plants that were transformed with the auxin reporter gene construct DR5::GUS. Hypergravity treatment at 300 g significantly increased ß-glucuronidase activity in inflorescence stems of DR5::GUS plants, indicating that endogenous auxin accumulation was enhanced by hypergravity treatment. The hypergravity-related increased expression levels of both DR5::GUS and lignin biosynthesis-related genes in inflorescence stems were suppressed after disbudding, indicating that the increased expression of lignin biosynthesis-related genes is dependent on an increase in auxin influx from the shoot apex.

Neuromedin U-induced anorexigenic action is mediated by the corticotropin-releasing hormone receptor-signaling pathway in goldfish.

Our recent research has indicated that neuromedin U (NMU) orthologs exist in goldfish, and that NMU consisting of 21 amino acid residues (NMU-21) can potently inhibit food intake in goldfish, as is the case in rodents. However, the anorexigenic pathway of NMU-21 has not yet been clarified in this species. Corticotropin-releasing hormone (CRH), CRH-related peptides and alpha-melanocyte-stimulating hormone (alpha-MSH), which exert potent anorexigenic effects, are important mediators involved in feeding regulation in fish. We examined whether CRH or alpha-MSH mediates NMU-21-induced anorexigenic action in goldfish. We first investigated the effect of intracerebroventricular (ICV) administration of NMU-21 at 100 pmol/g body weight (BW), which is enough to suppress food intake, on expression levels of mRNA for CRH and proopiomelanocortin (POMC) in the hypothalamus. ICV-injected NMU-21 induced a significant increase in the expression level of CRH mRNA, but not that of POMC mRNA. We also examined the effects of ICV administration of the CRH 1/2 receptor antagonist, alpha-helical CRH((9-41)), and the melanocortin 4 receptor antagonist, HS024, on the anorexigenic action of ICV-injected NMU-21. The anorexigenic effect of NMU-21 was blocked by treatment with alpha-helical CRH((9-41)) at 400 pmol/g BW, but not HS024 at 200 pmol/g BW. These results suggest that the anorexigenic action of NMU-21 is mediated by the CRH 1 or 2 receptor-signaling pathway in goldfish.

Localization and regulation of a facilitative urea transporter in the kidney of the red-eared slider turtle (Trachemys scripta elegans).

Urea is the major excretory end product of nitrogen metabolism in most chelonian reptiles. In the present study, we report the isolation of a 1632 base pair cDNA from turtle kidney with one open reading frame putatively encoding a 403-residue protein, the turtle urea transporter (turtle UT). The first cloned reptilian UT has high homology with UTs (facilitated urea transporters) cloned from vertebrates, and most closely resembles the UT-A subfamily. Injection of turtle UT cRNA into Xenopus oocytes induced a 6-fold increase in [(14)C]urea uptake that was inhibited by phloretin. The turtle UT mRNA expression and tissue distribution were examined by RT-PCR with total RNA from various tissues. Expression of turtle UT mRNA was restricted to the kidney, and no signal was detected in the other tissues, such as brain, heart, alimentary tract and urinary bladder. An approximately 58 kDa protein band was detected in membrane fractions of the kidney by western blot using an affinity-purified antibody that recognized turtle UT expressed in Xenopus oocytes. In an immunohistochemical study using the anti-turtle UT antibody, UT-immunopositive cells were observed along the distal tubule but not in the collecting duct. In turtles under dry conditions, plasma osmolality and urea concentration increased, and using semi-quantitative RT-PCR the UT mRNA expression level in the kidney was found to increase 2-fold compared with control. The present results, taken together, suggest that the turtle UT probably contributes to urea transport in the distal tubule segments of the kidney in response to hyperosmotic stress under dry conditions.

Cellular localization of a putative Na(+)/H (+) exchanger 3 during ontogeny in the pronephros and mesonephros of the Japanese black salamander (Hynobius nigrescens Stejneger).

The cloning of cDNA and an examination of the tissue distribution of Na(+)/H(+) exchanger 3 (NHE3) were carried out in the Japanese black salamander, Hynobius nigrescens. The cellular localization of Hynobius NHE3 was examined by in situ hybridization and immunohistochemistry during ontogeny in the nephron of the pronephros and mesonephros of the salamander. The partial amino acid sequence of Hynobius NHE3 was 81% and 72% identical to rat NHE3 and stingray NHE3, respectively. Hynobius NHE3 mRNA and protein were exclusively expressed along the late portion of the distal tubule to the anterior part of the pronephric duct of premetamorphic larvae (IY stages 43-50). NHE3 mRNA was expressed in the pronephros but not in the external gills in the larvae at the digit differentiation stage (IY stage 50). In the adult, mRNA was strongly expressed in the mesonephros but not in the ventral and dorsal skin. In juvenile and adult specimens, NHE3 immunoreactivity was observed at the apical membrane of the initial parts of the distal tubules of the mesonephric kidney. Immunohistochemical and in situ hybridization studies suggested that Na(+) absorption coupled with H(+) secretion via NHE3 occurred in the distal nephron of the pronephros and mesonephros. This is the first study to indicate NHE3 expression during ontogeny in amphibians.

Two RNA editing sites with cis-acting elements of moderate sequence identity are recognized by an identical site-recognition protein in tobacco chloroplasts.

The chloroplast genome of higher plants contains 20-40 C-to-U RNA editing sites, whose number and locations are diversified among plant species. Biochemical analyses using in vitro RNA editing systems with chloroplast extracts have suggested that there is one-to-one recognition between proteinous site recognition factors and their respective RNA editing sites, but their rigidness and generality are still unsettled. In this study, we addressed this question with the aid of an in vitro RNA editing system from tobacco chloroplast extracts and with UV-crosslinking experiments. We found that the ndhB-9 and ndhF-1 editing sites of tobacco chloroplast transcripts are both bound by the site-specific trans-acting factors of 95 kDa. Cross-competition experiments between ndhB-9 and ndhF-1 RNAs demonstrated that the 95 kDa proteins specifically binding to the ndhB-9 and ndhF-1 sites are the identical protein. The binding regions of the 95 kDa protein on the ndhB-9 and ndhF-1 transcripts showed 60% identity in nucleotide sequence. This is the first biochemical demonstration that a site recognition factor of chloroplast RNA editing recognizes plural sites. On the basis of this finding, we discuss how plant organellar RNA editing sites have diverged during evolution.

A simple in vitro RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of ndh transcripts.

RNA editing is found in various transcripts from land plant chloroplasts. In tobacco chloroplasts, C-to-U conversion occurs at 36 specific sites including two sites identified in this work. Our RNA editing assay system using chloroplast extracts facilitated biochemical analyses of editing reactions but required mRNAs labeled with (32)P at specific sites. Here, we have improved the in vitro system using fluorescence-labeled chain terminators, ddGTP and ddATP, and have measured the editing activity at 19 sites in ndh transcripts. Editing activities varied from site to site. It has been reported that one editing site in ndhA mRNAs is present in spinach but absent in tobacco, but a corresponding editing capacity had been found in vivo in tobacco using biolistic transformation. We confirmed biochemically the existence of this activity in tobacco extracts. Using the non-radioactive assay, we examined sequences essential for editing within a 50-nt mRNA region encompassing an editing site. Editing of the ndhB-2 site requires a short sequence in front of the editing site, while that of the ndhF mRNA requires two separate regions, a sequence surrounding the editing site and a 5' distal sequence. These results suggest that distinct editing mechanisms are present in chloroplasts.

Hypergravity stimulus enhances primary xylem development and decreases mechanical properties of secondary cell walls in inflorescence stems of Arabidopsis thaliana.

BACKGROUND AND AIMS: The xylem plays an important role in strengthening plant bodies. Past studies on xylem formation in tension woods in poplar and also in clinorotated Prunus tree stems lead to the suggestion that changes in the gravitational conditions affect morphology and mechanical properties of xylem vessels. The aim of this study was to examine effects of hypergravity stimulus on morphology and development of primary xylem vessels and on mechanical properties of isolated secondary wall preparations in inflorescence stems of arabidopsis. METHODS: Morphology of primary xylem was examined under a light microscope on cross-sections of inflorescence stems of arabidopsis plants, which had been grown for 3-5 d after exposure to hypergravity at 300 g for 24 h. Extensibility of secondary cell wall preparation, isolated from inflorescence stems by enzyme digestion of primary cell wall components (mainly composed of metaxylem elements), was examined. Plants were treated with gadolinium chloride, a blocker of mechanoreceptors, to test the involvement of mechanoreceptors in the responses to hypergravity. KEY RESULTS: Number of metaxylem elements per xylem, apparent thickness of the secondary thickenings, and cross-section area of metaxylem elements in inflorescence stems increased in response to hypergravity. Gadolinium chloride suppressed the effect of hypergravity on the increase both in the thickness of secondary thickenings and in the cross-section area of metaxylem elements, while it did not suppress the effect of hypergravity on the increase in the number of metaxylem elements. Extensibility of secondary cell wall preparation decreased in response to hypergravity. Gadolinium chloride suppressed the effect of hypergravity on cell wall extensibility. CONCLUSIONS: Hypergravity stimulus promotes metaxylem development and decreases extensibility of secondary cell walls, and mechanoreceptors were suggested to be involved in these processes.

Effects of hypergravity conditions on elongation growth and lignin formation in the inflorescence stem of Arabidopsis thaliana.

The effects of hypergravity on elongation growth and lignin deposition in secondary cell walls of the Arabidopsis thaliana (L.) Heynh. inflorescence stem were examined in plants grown for 3 days after exposure to hypergravity in the direction from shoot to root at 300 g for 24 h. The content of acetylbromide-extractable lignins in a secondary cell wall fraction prepared by enzyme digestion of inflorescence stem segments removing primary cell wall components was significantly increased by the hypergravity stimulus. Xylem vessels, particularly in a region closer to the base of the inflorescence stem, increased in number. Gadolinium chloride at 0.1 mM, a blocker of mechanoreceptors, partially suppressed the effect of hypergravity on lignin deposition in the secondary cell wall fraction. These results suggest that mechanoreceptors are responsible for hypergravity-induced lignin deposition in secondary cell walls in A. thaliana inflorescence stems.

Chloroplast phylogeny indicates that bryophytes are monophyletic.

Opinions on the basal relationship of land plants vary considerably and no phylogenetic tree with significant statistical support has been obtained. Here, we report phylogenetic analyses using 51 genes from the entire chloroplast genome sequences of 20 representative green plant species. The analyses, using translated amino acid sequences, indicated that extant bryophytes (mosses, liverworts, and hornworts) form a monophyletic group with high statistical confidence and that extant bryophytes are likely sisters to extant vascular plants, although the support for monophyletic vascular plants was not strong. Analyses at the nucleotide level could not resolve the basal relationship with statistical confidence. Bryophyte monophyly inferred using amino acid sequences has a good statistical foundation and is not rejected statistically by other data sets. We propose bryophyte monophyly as the currently best hypothesis.