Gibberellin induces alpha-amylase gene in seed coat of Ipomoea nil immature seeds

Two full-length cDNAs encoding gibberellin 3-oxidases, InGA3ox1 and InGA3ox2, were cloned from developing seeds of morning glory (Ipomoea nil (Pharbitis nil) Choisy cv. Violet) with degenerate-PCR and RACEs. The RNA-blot analysis for these clones revealed that the InGA3ox2 gene was organ-specifically expressed in the developing seeds at 6-18 days after anthesis. In situ hybridization showed the signals of InGA3ox2 mRNA in the seed coat, suggesting that active gibberellins (GAs) were synthesized in the tissue, although no active GA was detected there by immunohistochemistry. In situ hybridization analysis for InAmy1 (former PnAmy1) mRNA showed that InAmy1 was also synthesized in the seed coat. Both InGA3ox2 and InAmy1 genes were expressed spatially overlapped without a clear time lag, suggesting that both active GAs and InAmy1 were synthesized almost simultaneously in seed coat and secreted to the integument. These observations support the idea that GAs play an important role in seed development by inducing alpha-amylase.     

Crystal structure of the liganded anti-gibberellin A(4) antibody 4-B8(8)/E9 Fab fragment

Gibberellins, a class of plant hormones, consist of more than 120 members. Only a few of them are recognized by a receptor that remains unknown. The haptenic mouse monoclonal antibody, 4-B8(8)/E9, was generated against gibberellin A(4) (GA(4)) to recognize biologically active GA selectivity, and we attempted to confirm the binding properties between the antibody and GA(4). We carried out an X-ray crystallographic analysis of the 4-B8(8)/E9 Fab fragment complexed with GA(4) at a 2.8 A resolution by using the molecular replacement method. The crystal structure of the Fab fragment showed the typical immunoglobulin fold of the beta-barrel structure which is the common motif of all antibodies. A small hapten-combining site was made up of three heavy chain CDR loops. On the other hand, CDRs of the light chain did not interact directly with GA(4). The C/D rings of the GA(4) molecule were in van der Waals contact mainly with the aromatic side chain of Tyr100AH and Phe100BH of CDR-H3. The 3 beta-hydroxyl and 6 beta-carboxyl groups were, respectively, hydrogen-bonded to the main chain of Ala33H and to the Thr53H heavy chain.     

Highly sensitive quantitative analysis of nicotianamine using LC/ESI-TOF-MS with an internal standard

A highly sensitive quantitative method for analyzing nicotianamine (NA) by liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) is reported. Fluorenylmethoxycarbonylation of nicotianamine reduced its polarity and enabled its retention in a reversed-phase column. The adoption of N(epsilon)-nicotyllysine (NL) as an internal standard ensured reliable quantification by giving a linear calibration curve drawn between the NA/NL molar ratios of standard solutions injected and the NA/NL area ratios in mass chromatograms. The high sensitivity of this analytical method allowed us to measure the amount of NA. This analytical method has applications to all research concerning NA.     

The rice FISH BONE gene encodes a tryptophan aminotransferase,which affects pleiotropic auxin‐related processes

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole‐3‐pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole‐3‐acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin‐related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.     

Defense-related signaling by interaction of arabinogalactan proteins and beta-glucosyl Yariv reagent inhibits gibberellin signaling in barley aleurone cells

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at the plasma membrane and in extracellular spaces. A synthetic chemical, beta-glucosyl Yariv reagent (beta-GlcY), binds specifically to AGPs. We previously reported that gibberellin signaling is specifically inhibited by beta-GlcY treatment in barley aleurone protoplasts. In the present study, we found that beta-GlcY also inhibited gibberellin-induced programmed cell death (PCD) in aleurone cells. We examined the universality and specificity of the inhibitory effect of beta-GlcY on gibberellin signaling using microarray analysis and found that beta-GlcY was largely effective in repressing gibberellin-induced gene expression. In addition, >100 genes were up-regulated by beta-GlcY in a gibberellin-independent manner, and many of these were categorized as defense-related genes. Defense signaling triggered by several defense system inducers such as jasmonic acid and a chitin elicitor could inhibit gibberellin-inducible events such as alpha-amylase secretion, PCD and expression of some gibberellin-inducible genes in aleurone cells. Furthermore, beta-GlcY repressed the gibberellin-inducible Ca2+-ATPase gene which is important for gibberellin-dependent gene expression, and induced known repressors of gibberellin signaling, two WRKY genes and a NAK kinase gene. These effects of beta-GlcY were also phenocopied by the chitin elicitor and/or jasmonic acid. These results indicate that gibberellin signaling is under the regulation of defense-related signaling in aleurone cells. It is also probable that AGPs are involved in the perception of stimuli causing defense responses.     

Identification of Gibberellin A3 and Abscisic acid from Artemisia capillaris Leaves

The hypocholesterolemic effects of Lactobacillus acidophilus 43121 (43121) and a mixture of Lactobacillus casei and Bifidobacterium longum (MIX) were studied in hypercholesterolemia-induced pigs. Serum total cholesterol was decreased by supplementation of either 43121 or MIX, although, high-density lipoprotein cholesterol was not changed. The hypocholesterolemic effect of 43121 and MIX was mainly due to bile acid dehydroxylation, this effect being supplementation-time dependent.     

Isolation and Characterization of Glucosyl Esters of Gibberellin A5 and A44 from Immature Seeds of Pharbitis purpurea

A method for predicting the enthalpy profile during extrusion cooking was developed. Wattmeters were connected to each barrel, and the heat loss from the barrels was measured under several temperature patterns without any material flow through the twin-screw extruder. A short section of some screws was replaced by a collar to make it possible to insert thermometers into the barrels during extrusion, and water was then pumped into the extruder while heating the barrels and the die. The measured temperature coincided with the temperature calculated from the consumed energy and the heat loss from each barrel, indicating that the enthalpy profile could be evaluated by this method. As an application of the method, the dependence of the enthalpy profile on the screw configuration was investigated. The enthalpy absorbed by the extruded materials was increased by using reverse screws and a needle valve at the outlet of the twin-screw extruder. These results suggest that the method developed in the present study will contribute to the design of extruder screws.     

Qualitative and semi-quantitative analyses of cytokinins using LC/APCI-MS in combination with ELISA

Application of liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) for the analysis of cytokinins was examined. The fragmentation of cytokinins was studied using authentic trans-zeatin (t-Z), trans-zeatin riboside (t-ZR), isopentenyl adenine (i⁶Ade), isopentenyl adenosine (i⁶Ado), benzyl adenine (BAde), benzyl adenosine (BAdo), and kinetin. These cytokinins were effectively ionized by APCI in aqueous acetonitrile. t-Z, i⁶Ade, BAde, and kinetin showed prominent quasi-molecular ions of [M + H]⁺, and ribosylcytokinins clearly showed both [M + H]⁺ and a characteristic fragment ion ([M + H-ribose]⁺), giving some information about their structures. The qualitative and semi-quantitative analyses of cytokinins by LC/APCI-MS were validated in combination with enzyme-linked immunosorbent assay (ELISA) through the analysis of t-ZR in the teratoma of Nicotiana tobacum. t-ZR was conclusively identified and a semi-quantitative estimate of its endogenous levels were provided by the combination of LC/APCI-MS and ELISA. The quantified values obtained by LC/ APCI-MS (single ion detection) and ELISA are in close agreement.