The subcellular localization of two isopentenyl diphosphate isomerases in rice suggests a role for the endoplasmic reticulum in isoprenoid biosynthesis.

KEY MESSAGE: Both OsIPPI1 and OsIPPI2 enzymes are found in the endoplasmic reticulum, providing novel important insights into the role of this compartment in the synthesis of MVA pathway isoprenoids. Isoprenoids are synthesized from the precursor's isopentenyl diphosphate (IPP) and dimethylallyl diphosphosphate (DMAPP), which are interconverted by the enzyme isopentenyl diphosphate isomerase (IPPI). Many plants express multiple isoforms of IPPI, the only enzyme shared by the mevalonate (MVA) and non-mevalonate (MEP) pathways, but little is known about their specific roles. Rice (Oryza sativa) has two IPPI isoforms (OsIPPI1 and OsIPPI2). We, therefore, carried out a comprehensive comparison of IPPI gene expression, protein localization, and isoprenoid biosynthesis in this species. We found that OsIPPI1 mRNA was more abundant than OsIPPI2 mRNA in all tissues, and its expression in de-etiolated leaves mirrored the accumulation of phytosterols, suggesting a key role in the synthesis of MVA pathway isoprenoids. We investigated the subcellular localization of both isoforms by constitutively expressing them as fusions with synthetic green fluorescent protein. Both proteins localized to the endoplasmic reticulum (ER) as well as peroxisomes and mitochondria, whereas only OsIPPI2 was detected in plastids, due to an N-terminal transit peptide which is not present in OsIPPI1. Despite the plastidial location of OsIPPI2, the expression of OsIPPI2 mRNA did not mirror the accumulation of chlorophylls or carotenoids, indicating that OsIPPI2 may be a redundant component of the MEP pathway. The detection of both OsIPPI isoforms in the ER indicates that DMAPP can be synthesized de novo in this compartment. Our work shows that the ER plays an as yet unknown role in the synthesis of MVA-derived isoprenoids, with important implications for the metabolic engineering of isoprenoid biosynthesis in higher plants.

Cloning and functional analysis of the promoters that upregulate carotenogenic gene expression during flower development in Gentiana lutea.

Over the last two decades, many carotenogenic genes have been cloned and used to generate metabolically engineered plants producing higher levels of carotenoids. However, comparatively little is known about the regulation of endogenous carotenogenic genes in higher plants, and this restricts our ability to predict how engineered plants will perform in terms of carotenoid content and composition. During petal development in the Great Yellow Gentian (Gentiana lutea), carotenoid accumulation, the formation of chromoplasts and the upregulation of several carotenogenic genes are temporally coordinated. We investigated the regulatory mechanisms responsible for this coordinated expression by isolating five G. lutea carotenogenic gene (GlPDS, GlZDS, GlLYCB, GlBCH and GlLYCE) promoters by inverse polymerase chain reaction (PCR). Each promoter was sufficient for developmentally regulated expression of the gusA reporter gene following transient expression in tomato (Solanum lycopersicum cv. Micro-Tom). Interestingly, the GlLYCB and GlBCH promoters drove high levels of gusA expression in chromoplast-containing mature green fruits, but low levels in chloroplast-containing immature green fruits, indicating a strict correlation between promoter activity, tomato fruit development and chromoplast differentiation. As well as core promoter elements such as TATA and CAAT boxes, all five promoters together with previously characterized GlZEP promoter contained three common cis-regulatory motifs involved in the response to methyl jasmonate (CGTCA) and ethylene (ATCTA), and required for endosperm expression (Skn-1_motif, GTCAT). These shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation. Our data provide insight into the regulatory basis of the coordinated upregulation of carotenogenic gene expression during flower development in G. lutea.

Recent Progress of the Practical Applications of the Platinum Nanoparticle-Based Electrochemistry Biosensors.

The detection of biomolecules using various biosensors with excellent sensitivity, selectivity, stability, and reproducibility, is of great significance in the analytical and biomedical fields toward achieving their practical applications. Noble metal nanoparticles are favorable candidates due to their unique optical, surface electrical effect, and catalytic properties. Among these noble metal nanoparticles, platinum nanoparticles (Pt NPs) have been widely employed for the detection of bioactive substances such as glucose, glutamic acid, and hormones. However, there is still a long way to go before the potential challenges in the practical applications of biomolecules are fully overcome. Bearing this in mind, combined with our research experience, we summarized the recent progress of the Pt NP-based biosensors and highlighted the current problems that exist in their practical applications. The current review would provide fundamental guidance for future applications using the Pt NP-based biosensors in food, agricultural, and medical fields.

ß-Actin facilitates etoposide-induced p53 nuclear import.

As a tumor suppressor, p53 preserves genomic integrity in eukaryotes. However, limited evidence is available for the p53 shuttling between the cytoplasm and nucleus. Previous studies have shown that ß-actin polymerization negatively regulates p53 nuclear import through its interaction with p53. In this study, we found that DNA damage induces both ß-actin and p53 accumulation in the nucleus. ß-actin knockdown impaired the nuclear transport of p53. Additionally, ß-actin could interact with p53 which was enhanced in response to genotoxic stress. Furthermore, N terminal deletion mutants of p53 shows reduced levels of association with ß-actin. We further identified Ser15, Thr18 and Ser20 of p53 are critical to the ß-actin: p53 interaction, which upon mutation into alanine abrogates the binding. Taken together, this study reveals that ß-actin regulates the nuclear import of p53 through protein-protein interaction.

Red Anthocyanins and Yellow Carotenoids Form the Color of Orange-Flower Gentian (Gentiana lutea L. var. aurantiaca).

Flower color is an important characteristic that determines the commercial value of ornamental plants. Gentian flowers occur in a limited range of colors because this species is not widely cultivated as a cut flower. Gentiana lutea L. var. aurantiaca (abbr, aurantiaca) is characterized by its orange flowers, but the specific pigments responsible for this coloration are unknown. We therefore investigated the carotenoid and flavonoid composition of petals during flower development in the orange-flowered gentian variety of aurantiaca and the yellow-flowered variety of G. lutea L. var. lutea (abbr, lutea). We observed minor varietal differences in the concentration of carotenoids at the early and final stages, but only aurantiaca petals accumulated pelargonidin glycosides, whereas these compounds were not found in lutea petals. We cloned and sequenced the anthocyanin biosynthetic gene fragments from petals, and analyzed the expression of these genes in the petals of both varieties to determine the molecular mechanisms responsible for the differences in petal color. Comparisons of deduced amino acid sequences encoded by the isolated anthocyanin cDNA fragments indicated that chalcone synthase (CHS), chalcone isomerase (CHI), anthocyanidin synthase 1 (ANS1) and ANS2 are identical in both aurantiaca and lutea varieties whereas minor amino acid differences of the deduced flavonone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) between both varieties were observed. The aurantiaca petals expressed substantially higher levels of transcripts representing CHS, F3H, DFR, ANS and UDP-glucose:flavonoid-3-O-glucosyltransferase genes, compared to lutea petals. Pelargonidin glycoside synthesis in aurantiaca petals therefore appears to reflect the higher steady-state levels of pelargonidin synthesis transcripts. Moreover, possible changes in the substrate specificity of DFR enzymes may represent additional mechanisms for producing red pelargonidin glycosides in petals of aurantiaca. Our report describing the exclusive accumulation of pelargonidin glycosides in aurantiaca petals may facilitate the modification of gentian flower color by the production of red anthocyanins.

Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene.

The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity.

Effects of glycoalkaloids from Solanum plants on cucumber root growth.

The phytotoxic effect of four glycoalkaloids and two 6-O-sulfated glycoalkaloid derivatives were evaluated by testing their inhibition of cucumber root growth. The bioassays were performed using both compounds singly and in equimolar mixtures, respectively. Cucumber root growth was reduced by chaconine (C), solanine (S), solamargine (SM) and solasonine (SS) with IC(50) values of 260 (C), 380 (S), 530 (SM), and 610 microM (SS). The inhibitory effect was concentration-dependent. 6-O-sulfated chaconine and 6-O-sulfated solamargine had no inhibitory effects, which indicated that the carbohydrate moieties play an important role in inhibiting cucumber root growth. The equimolar mixtures of paired glycoalkaloids, both chaconine/solanine and solamargine/solasonine, produced synergistic effects on inhibition of cucumber root growth. By contrast, mixtures of unpaired glycoalkaloids from different plants had no obviously synergistic effects. The growth inhibited plant roots lacked hairs, which implied that inhibition was perhaps at the level of root hair growth.

A novel water-soluble beta-(1-->6)-D-glucan isolated from the fruit bodies of Bulgaria inquinans (Fries).

A low molecular-weight polysaccharide named BIWP2 was purified from the fruit bodies of Bulgaria Inquinans (Fries) via hot-water extraction, followed by freeze-thawing and gel filtration chromatography on Sephadex G-75. Monosaccharide composition analysis revealed that BIWP2 contained exclusively glucose. High performance size exclusion chromatography (HPSEC) showed that it was a homogeneous polysaccharide fraction. Its molecular weight was estimated to be 2.6 KD and the polydispersity index (M(w)/M(n)) was calculated to be 1.4. Periodate oxidation, methylation, and NMR analyses indicated that BIWP2 was a linear beta-(1-->6)-D-glucan without side chains. This is the first time to report a linear beta-(1-->6)-D-glucan with low molecular weight in non-lichenized ascomycete.