Nectaries are secretory organs that are widely present in flowering plants that function to attract floral pollinators. Owing to diversity in nectary positions and structures, they are thought to have originated multiple times during angiosperm evolution, with their potential contribution to the diversification of flowering plants and pollinating animals being considerable. We investigated the genetic basis of diverse nectary forms in eudicot angiosperm species using CRABS CLAW (CRC), a gene required for nectaries in Arabidopsis. CRC expression is conserved in morphologically different nectaries from several core eudicot species and is required for nectary development in both rosids and asterids,two major phylogenetic lineages of eudicots. However, in a basal eudicot species, no evidence of CRC expression in nectaries was found. Considering the phylogenetic distribution of nectary positions and CRC expression analyses in eudicots, we propose that diverse nectaries in core eudicots share conserved CRC gene regulation, and that derived nectary positions in eudicots have altered regulation of CRC. As the ancestral function of CRC lies in the regulation of carpel development, it may have been co-opted as a regulator of nectary development within the eudicots, concomitant with the association of nectaries with reproductive organs in derived lineages.

Agrobacterium-mediated infection of petunia (Petunia hybrida) plants with tobacco rattle virus (TRV) bearing fragments of Petunia genes resulted in systemic infection and virus-induced gene silencing (VIGS) of the homologous host genes. Infection with TRV containing a phytoene desaturase (PDS) fragment resulted in reduced abundance of PDS transcripts and typical photobleaching of photosynthetic tissues. Infection with TRV containing a chalcone synthase (CHS) fragment resulted in silencing of anthocyanin production in infected flowers. The silencing phenotype ranged from scattered white spots on the normal purple background to entirely white flowers. Symptoms in the V26 cultivar were a diffuse mosaic, but infection of some purple-flowered commercial cultivars resulted in large white sectors and even entirely white flowers. Abundance of CHS transcripts in the white flowers was less than 4% of that in purple flowers on the same plant. Infection with TRV containing a tandem construct of PDS and CHS resulted in leaf photobleaching and white patterns on the flowers. Transcripts of CHS and PDS were reduced both in leaves and in flowers confirming simultaneous silencing of both genes by the tandem construct. We tested the effects of infection with TRV containing CHS and a fragment of a petunia gene encoding for 1-aminocyclopropane-1-carboxylate oxidase (ACO4) Abundance of transcripts encoding ACO4 and ACO1 were reduced (by 5% and 20%, respectively) in infected flowers. Whether the flowers were treated with ACC or pollinated, the white (silenced) flowers or flower sectors produced less ethylene and senesced later than purple (non-silenced) tissues. These results indicate the value of VIGS with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of floral-associated genes.

SummaryProhibitins, highly conserved mitochondrial proteins, have been shown to play important roles in cell cycling and senescence in animals and yeast. Sequences with high similarity to prohibitins have been identified in a number of plant species, but their function has not yet been demonstrated. The deduced amino acid sequences of PhPHB1 and PhPHB2, sequences that we identified in a petunia floral expressed sequence tag (EST) database, show high similarity to those of prohibitin-1 and prohibitin-2 proteins, respectively, reported from yeast, animals and plants. Southern analysis suggested that these genes were members of small gene families with at least two prohibitin-1 homologs and four prohibitin-2 homologs. When we downregulated expression of prohibitin-1 using a Tobacco rattle virus-based (TRV), virus-induced gene silencing system (VIGS), we observed plants with smaller and distorted leaves and flowers. Cells in silenced flowers were larger than in control flowers, indicating a substantial reduction in the number of cell divisions that took place during corolla development. The life of silenced flowers was shorter than that of controls, whether on the plant or detached. The respiration of silenced flowers was higher than that of controls, and we observed a marked increase in the abundance of transcripts of a catalase and a small heat-shock protein in the silenced flowers. Our data indicate that prohibitins play a key role in plant development and senescence.

95616 (J.-C.C., M.S.R.)To understand how auxin regulates sensitivity of abscission zone (AZ) tissues to ethylene, we used a polymerase chain reactionbased subtractive approach to identify gene transcripts in Mirabilis jalapa AZs that changed in abundance during the time the zones became competent to abscise in response to exogenous ethylene. Transcript expression was then examined in leaf and stem AZs over the period they became ethylene competent following indole-3-acetic acid (IAA) depletion either by leaf deblading, treatment with the IAA transport inhibitor naphthylphthalamic acid, or cutting the stem above a node (decapitation). Transcripts down-regulated by deblading/decapitation included Mj-Aux/IAA1 and Mj-Aux/IAA2, encoding Aux/IAA proteins, and three other transcripts showing highest identity to a polygalacturonase inhibitor protein, a b-expansin, and a b-tubulin. Application of IAA to the cut end of petioles or stumps inhibited abscission, and prevented the decline in the levels of transcripts in both AZs. Transcripts up-regulated in the AZ following deblading/decapitation or treatment with naphthylphthalamic acid were isolated from plants pretreated with 1-methylcyclopropene before deblading to help select against ethylene-induced genes. Some of the up-regulated transcripts showed identity to proteins associated with ethylene or stress responses, while others did not show homology to known sequences. Sucrose infiltration of stem stumps enhanced abscission following ethylene treatment and also enhanced the induction of some of the up-regulated genes. Our results demonstrate a correlation between acquisition of competence to respond to ethylene in both leaf and stem AZs, and decline in abundance of auxin regulatory gene transcripts.

Widely distributed bottom simulating reflectors (BSRs) imply the potential existence of gas hydrates offshore southwestern Taiwan. To compare the distribution of methane concentrations along passive and active margins in the region, bottom waters and cored sediments were collected during four cruises from 2005 to 2006. The results reveal that sites with high methane concentrations are predominantly distributed in the active margin and site GS5 is the only site that contains very high methane concentrations in the passive margin of studied area. Anomalously high methane fluxes still can be obtained from the calculation of diffusive methane flux, although there might be some gas leakage during or after sampling procedures. The profiles of methane and sulfate concentration reveal very shallow depths of the sulfate-methane interface (SMI) at some sites. There is evidence that sulfate reduction is mainly driven by the process of anaerobic methane oxidation. Thus, sulfate fluxes can be used as a proxy for methane fluxes through the use of diffusion equations; and the results show that the fluxes are very high in offshore southwestern Taiwan. The depths of the SMI are different at sites GH6 and C; however, both methane profiles reveal parallel methane gradients below the SMI. This might be because of methane migration to surface sediments from the same reservoir with the same diffusion rates. Although BSRs are widely distributed both in the active margin and in the passive margin, most sites with high methane concentrations have been found in the active margin. Therefore, the specific tectonic settings in offshore SW Taiwan might strongly control the stability of gas hydrates, and thus affect the methane concentrations and fluxes of the sediments and sea waters. Furthermore, the carbon isotopic composition of methane shows that a biogenic gas source is dominant at shallower depth; however, some thermogenic gases might be introduced through the fracture ⁄ fault zones from deeper source in the active region of studied area.

When the abundance of the FOX1 gene product is reduced, Chlamydomonas cells grow poorly in iron-deficient medium, but not in iron-replete medium, suggesting that FOX1-dependent iron uptake is a high-affinity pathway. Alternative pathways for iron assimilation, such as those involving ZIP family transporters IRT1 and IRT2, may be operational.Iron is an essential nutrient for all forms of life because of its role as a catalyst in redox reactions that are the basis of life. Yet, because of the reduced solubility of ferric salts (the predominant species in the aerobic world), particularly in an alkaline soil environment, the bioavailability of iron can be poor and, hence, a limitation to life (6). Accordingly, the assimilation pathways include mechanisms for mobilizing iron from inaccessible forms, plus mechanisms for uptake.Previously, we and others identified several genes that might constitute an iron uptake pathway in Chlamydomonas based on their increased expression in iron-poor growth conditions and/or on their sequence relationship to components of iron assimilation in fungi, plants, or animals (1, 14). These include FRE1, encoding a ferrireductase; FOX1, encoding a putative multicopper oxidase; FTR1, encoding a ferric transporter; FEA1/2, encoding abundant periplasmic proteins; and IRT1/2, encoding ZIP-family transporters. FRE1 and the IRT proteins are most-closely related to components found in the iron assimilation pathway of plants; FTR1-like proteins are found primarily in the fungi, although a homolog does occur in Physcomitrella; the FEA proteins are only in algae and a dinoflagellate; and FOX1 is a domain-rearranged version of animal ferroxidases, like ceruloplasmin and hephaestin, that function in iron homeostasis (1,5,14,21,23). Except for the FEA proteins, the evidence for the participation of these components in iron assimilation in Chlamydomonas is indirect. Furthermore, the role of a FOX1/FTR1-type transporter (analogous to fungal iron uptake components) versus that of the IRT-type transporter is not clear. In Saccharomyces cerevisiae, there are multiple pathways that together participate in iron uptake, so that the organism can use a variety of iron sources supplied at a range of concentrations (reviewed in references 13 and 19).FOX1 is localized to the plasma membrane of Chlamydomonas; its sequence indicates that it is a multicopper oxidase, and therefore, by analogy to S. cerevisiae Fet3p, FOX1 is proposed to oxidize ferrous to ferric iron for uptake by FTR1 (9, 14, 24). In S. cerevisiae and in animals, nutritional copper deficiency or genetic defects that result in copper deficiency have an impact also on iron nutrition because of the function of the multicopper oxidases, Fet3p and ceruloplasmin, in iron metabolism (reviewed in references 2, 7, and 11). Yet in our previous work, we have found that copper-deficient wild-type Chlamydomonas cells are not iron-deficient even when iron is supplied at nutritionally limiting concentrations (14). Nevertheless, this is not the case in the crd2 mutant...

Rhodopseudomonas palustris strain PS3, a phototrophic bacterium, was originally isolated from a paddy field located in Taipei city, Taiwan, and showed positive effects on the growth of leafy vegetables. The aim of this study was to clarify the mechanism of the beneficial effects exerted by PS3 on plants. An ineffective R. palustris strain, YSC3, isolated from a paddy field located in Yilan County, was used as the negative control for comparative analyses. We cultivated non-heading Chinese cabbage (Brassica rapa var. chinensis) in 1/2 strength Hoagland hydroponic solution, in which nitrate is the main nitrogen source. We evaluated various plant physiological responses to inoculation with different bacterial inoculants. The N use efficiency (NUE) of PS3-inoculated plants was dramatically higher than that of YSC3-inoculated plants. The nitrate uptake efficiency (NUpE) was significantly elevated in plants treated with PS3; however, no excess nitrate accumulation was observed in leaves. We also noticed that the endogenous indole-3-acetic acid (IAA) levels as well as the cell division rate in the leaves of PS3-inoculated plants were significantly higher than those in the leaves of YSC3-inoculated plants. We examined the bacterial transcription of some genes during root colonization, and found that the expression level of IAA synthesis related gene MAO was almost the same between these two strains. It suggests that the elevated endogenous IAA in the PS3-inoculated plants was not directly derived from the exogenous IAA produced by this bacterium. Taken together, we deduced that PS3 inoculation could promote plant growth by enhancing nitrate uptake and stimulating the accumulation of endogenous auxin in young expanding leaves to increase the proliferation of leaf cells during leaf development.

The genetic regulatory mechanisms that govern natural corolla senescence in petunia are not well understood. To identify key genes and pathways that regulate the process, we performed a transcriptome analysis in petunia corolla at four developmental stages, including corolla fully opening without anther dehiscence (D0), corolla expansion, 2 days after anthesis (D2), corolla with initial signs of senescence (D4), and wilting corolla (D7). We identified large numbers of differentially expressed genes (DEGs), ranging from 4626 between the transition from D0 and D2, 1116 between D2 and D4, a transition to the onset of flower senescence, and 327 between D4 and D7, a developmental stage representing flower senescence. KEGG analysis showed that the auxin- and ethylene-related hormone biosynthesis and signaling transduction pathways were significantly activated during the flower development and highly upregulated at onset of flower senescence. Ethylene emission was detected at the D2 to D4 transition, followed by a large eruption at the D4 to D7 transition. Furthermore, large numbers of transcription factors (TFs) were activated over the course of senescence. Functional analysis by virus-induced gene silencing (VIGS) experiments demonstrated that inhibition of the expression of TFs, such as ethylene-related ERF, auxin-related ARF, bHLH, HB, and MADS-box, significantly extended or shortened flower longevity. Our data suggest that hormonal interaction between auxin and ethylene may play critical regulatory roles in the onset of natural corolla senescence in petunia.