Enhanced benzophenanthridine alkaloid production and protein expression with combined elicitor in <Emphasis Type="Italic">Eschscholtzia californica</Emphasis> suspension cultures

Production of the benzophenanthridine alkaloids in Eschscholtzia californica suspension cell cultures was optimized by adding 0.5 mg methyl jasmonate (MJ) and 0.02 mg salicylic acid (SA)/g FCW after 7 days cultivation. Sanguinarine reached 24 mg/g DCW by such treatment; 10 times higher than in control cell cultures. MJ and SA induced expression of berberine bridge enzyme and 3′-hydroxy-(S)-N-methylcoclaurine-4′-O-methyltransferase, respectively. MJ plus SA induced over-expression of both enzymes.     

In vitro induction of inflorescence in <Emphasis Type="Italic">Dioscorea</Emphasis><Emphasis Type="Italic">zingiberensis</Emphasis>

Inflorescence induction and morphogenesis of regenerated flowers were investigated in vitro in Dioscorea zingiberensis C. H. Wright. Inflorescence induction was influenced by the type and concentration of phytohormones. When floral bud explants were incubated on a Murashige and Skoog medium containing a combination of 2.0 mg l⁻¹ 6-benzyladenine and 0.5 mg l⁻¹ indole-3-butyric acid, the highest frequency of inflorescence induction was observed. However, in the presence of gibberellic acid, induction efficiency was reduced although node length of inflorescence was increased. Ontogenetic studies revealed that the inflorescence primordia originated directly from axillary epidermal cells of the perianth and bract of the explants after 7 days. In vitro, male flowers developed normally and blossomed after 90–100 days. In addition, some bisexual flowers were observed. These results demonstrated that there were differences in sexual differentiation of floral buds in vitro compared with that in vivo.     

A new pathogenesis-related protein,LrPR4, from <Emphasis Type="Italic">Lycoris radiata</Emphasis>, and its antifungal activity against <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

A new Lycoris radiata pathogenesis-related (PR)-4 gene, LrPR4 was isolated. LrPR4 encodes a 142 amino acid protein with a predicted molecular mass of 15.43 kDa and pI of 7.56. The putative LrPR4 shows high similarity to PR4 type proteins from various plant species and belongs to the Barwin family. Like other PR4s from monocot plants, LrPR4 protein contains a conserved Barwin domain and has a signal peptide at its N-terminus. The recombinant LrPR4 protein expressed in Escherichia coli showed activity towards hydrolysing RNA from L. radiata bulbs and antifungal activity. The results of this study suggest that LrPR4 may play a role in the disease resistance responses of plant against pathogen attacks though its antifungal activity.     

Identification and expression analysis of <Emphasis Type="Italic">CjLTI</Emphasis>, a novel low temperature responsive gene from <Emphasis Type="Italic">Caragana jubata</Emphasis>

Using rapid amplification of cDNA ends, a full length cDNA (CjLTI) was cloned from apical buds of Caragana jubata, a plant species that grows under extreme cold. The cDNA obtained was 573 bp long consisting of an open reading frame of 351 bp encoding 116 amino acids. Homology analysis did not exhibit significant similarity with any sequence at NCBI database, therefore it was deduced as a novel gene. Secondary structure analysis suggested that the deduced CjLTI contained 25.86% α-helices, 4.31% β-turns, 6.90% extended strands, and 62.93% random coils. The hydropathy profile suggested CjLTI to be a hydrophobic protein having characteristic features of signal peptides at N-terminus. The gene exhibited down-regulation at 5 min of exposure to low temperature (LT, 4 ± 3°C) followed by a strong up-regulation after 15 min and onwards. Methyl jasmonate (MJ) lead to up-regulation of CjLTI starting at 5 min onwards. The gene exhibited up- and down-regulation of expression pattern in response to abscisic acid (ABA) and salicylic acid (SA). Mild drought stress slightly up-regulated gene expression and at severe drought (up to 115% reduction in leaf water potential) slight down-regulation of gene expression was observed. These results suggested CjLTI to be a LT responsive gene wherein MJ, ABA and SA pathways might be involved in regulating the gene expression.     

Effect of salicylic acid and methyl jasmonate on antioxidant systems of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>

The influence of phytohormones, salicylic acid (SA) and methyl jasmonate (MJ) on the antioxidant systems in Haematococcus pluvialis was investigated. Both SA and MJ at 500 μM concentration reduced the growth of alga with salicylic acid, having more pronounced effect. Carotenoid and chlorophyll contents were decreased by SA and increased by MJ. Salicylic acid (100 μM) increased astaxanthin content to 6.8-fold under low light (30 μmol m⁻² s⁻¹), while MJ (10 μM) showed marginal increase in astaxanthin. Salicylic acid (500 μM) increased superoxide dismutase activity to 4.5- and 3.3-fold and ascorbate peroxidase (APX) activity to 15.5- and 7.1-fold under low and high light, respectively. Methyl jasmonate increased catalase activity (1.4-fold) under high light and APX activity (5.4-fold) under low light. Different mechanism of oxidative stress induced antioxidant production may be the plausible reason for this varied response for salicylic acid and methyl jasmonate. Higher concentrations of SA and MJ inhibited astaxanthin accumulation by different mechanisms either by scavenging the free radicals or by increasing primary carotenoids production. At lower concentrations, these phytohormones could be used for elicitation of secondary carotenoid production.     

Cloning and characterization of truncated <Emphasis Type="Italic">cry1Ab</Emphasis> gene from a new indigenous isolate of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The insecticidal crystal protein(s) encoded by cry gene(s) of Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. A new 3′-truncated cry1Ab gene was cloned from an indigenous isolate of Bt, A19-31. Nucleotide sequencing and homology search revealed that the deduced amino acid sequence of Cry1Ab toxin of Bt strain A19-31 had a variation of two amino acid residues with the holotype sequence, Cry1Ab1. Expression of the 3′-truncated cry1Ab gene was studied in an acrystalliferous strain of Bt (4Q7). SDS-PAGE and immunostrip analysis of spore-crystal mixture revealed a low level expression of the 3′-truncated cry1Ab gene. Insecticidal activity assay showed that the recombinant 3′-truncated cry1Ab gene product was toxic to larvae of both Helicoverpa armigera and Spodoptera litura.     

New species of <Emphasis Type="Italic">Chalybea</Emphasis> and <Emphasis Type="Italic">Huilaea</Emphasis> (Melastomataceae)

One new species each is proposed in Chalybea and Huilaea (Melastomataceae: Blakeeae). Chalybea peruviana has elliptic, 5-plinerved leaves with entire, revolute margins, inflorescences with 33–39 flowers, and is endemic to Peru. Huilaea calyptrata has inflorescences with 15–17, irregularly calyptrate flowers, anthers with a warty connective in the shape of an inverted hand fan, and is endemic to Ecuador. A key to the eight species of Huilaea is provided.     

Strong Expression and Conserved Regulation of <Emphasis Type="Italic">ACT2</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Arabidopsis ACT2 represents an ancient class of vegetative plant actins and is strongly and constitutively expressed in almost all Arabidopsis sporophyte vegetative tissues. Using the beta glucuronidase report system, the studies showed that ACT2 5′ regulatory region was significantly more active than CaMV 35S promoter in Arabidopsis seedlings and gametophyte vegetative tissues of Physcomitrella patens. Its activity was also observed in rice and maize seedlings. Thus, the ACT2 5′ regulatory region could potentially serve as a strong regulator to express a transgene in divergent plant species. ACT2 5′ regulatory region contained 15 conserved sequence elements, an ancient intron in its 5′ un-translated region (5′ UTR), and a purine-rich stretch followed by a pyrimidine-rich stretch (PuPy). Mutagenesis and deletion analysis illustrated that some of the conserved sequence elements and the region containing PuPy sequences played regulatory roles in Arabidopsis. Interestingly, mutation of the conserved elements did not lead a dramatic change in the activity of ACT2 5′ regulatory region. The ancient intron in ACT2 5′ UTR was required for its strong expression in both Arabidopsis and P. patens, but did not fully function as a canonical intron. Thus, it was likely that some of the conserved sequence elements and gene structures had been preserved in ACT2 5′ regulatory region over the course of land plant evolution partly due to their functional importance. The studies provided additional evidences that identification of evolutionarily conserved features in non-coding region might be used as an efficient strategy to predict gene regulatory elements.     

Purification and properties of aminoaldehyde dehydrogenase from <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5′-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS–PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a pI value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that Avena AMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K _m values for APAL, ABAL, and GBAL were 1.5×10^–6, 2.2×10^–6, and 1.3×10^–5 M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH. Electronic Publication     

Photosynthetic performance of <Emphasis Type="Italic">Lycoris radiata</Emphasis> var. <Emphasis Type="Italic">radiata</Emphasis> to shade treatments

The effects of shade on the growth, leaf photosynthetic characteristics, and chlorophyll (Chl) fluorescence parameters of Lycoris radiata var. radiata were determined under differing irradiances (15, 65, and 100% of full irradiance) within pots. The HI plants exhibited a typical decline in net photosynthetic rate (P _N) during midday, which was not observed in MI- and LI plants. This indicated a possible photoinhibition in HI plants as the ratio of variable to maximum fluorescence (F_v/F_m) value was higher and the minimal fluorescence (F₀) was lower in the, and LI plants. Diurnal patterns of stomatal conductance (g _s) and transpiration rate (E) were remarkably similar to those of P _N at each shade treatments, and the intercellular CO₂ concentration (C _i) had the opposite change trend. Under both shading conditions, the light saturation point, light compensation point and photon-saturated photosynthetic rate (P _max) became lower than those under full sunlight, and it was the opposite for the apparent quantum yield (AQY). The higher the level of shade, the lower the integrated daytime carbon gain, stomatal and epidermis cell densities, specific leaf mass (SLM), bulb mass ratio (BMR), leaf thickness, and Chl a/b ratio. In contrast, contents of Chls per dry mass (DM), leaf area ratio (LAR), leaf mass ratio (LMR), leaf length, leaf area and total leaf area per plant increased under the same shade levels to promote photon absorption and to compensate for the lower radiant energy. Therefore, when the integrated daytime carbon gain, leaf area and total leaf area per plant, which are the main factors determining the productivity of L. radiata var. radiata plant, were taken into account together, this species may be cultivated at about 60∼70% of ambient irradiance to promote its growth.     

Molecular cloning and characterization of the promoter of <Emphasis Type="Italic">SmGGPPs</Emphasis> and its expression pattern in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. It regulates the formation of diterpenoid, such as tanshinones. We cloned a gene for GGPP synthase SmGGPPs involved in diterpenoid biosynthesis from Salvia miltiorrhiza. At 2,767 bp long, this gene comprises an intron and two exons that encode a polypeptide of 364 amino acid residues. Then the 5′ flanking sequence of SmGGPPs was characterized by bioinformatics method. Deletion analysis of the promoter of SmGGPPs using tobacco plant displayed that the promoter was induced by heat and cold. To further search these cis-elements involved in induction regulation in the 5′ flanking sequence of SmGGPPs, many putative cis-elements were predicted with the PlantCARE and PLACE databases. A group of putative cis-acting elements are involved in induction regulation, including G-Box, WRKY, MYC and ATCT motifs. Real-time PCR analysis revealed that SmGGPPs is mainly expressed in the leaves and can also be induced by various factors, such as NaCl, wounding, high temperature, darkness, pathogen, methyl jasmonate, abscisic acid, salicylic acid, and gibberellins. This study provides useful information for further study of SmGGPPs and its regulator effect on the biosynthetic process of tanshinones so that researchers can improve the tanshinone contents in S. miltiorrhiza.     

Aminoglycoside-Resistance Mechanisms in Multidrug-Resistant <Emphasis Type="Italic">Staphylococcus </Emphasis><Emphasis Type="Italic">aureus</Emphasis> Clinical Isolates

Aminoglycoside resistance in six clinically isolated Staphylococcus aureus was evaluated. Genotypical examination revealed that three isolates (HLGR-10, HLGR-12, and MSSA-21) have aminoglycoside-modifying enzyme (AME) coding genes and another three (GRSA-2, GRSA-4, and GRSA-6) lacked these genes in their genome. Whereas isolates HLGR-10 and HLGR-14 possessed bifunctional AME coding gene aac(6′)-aph(2′′), and aph(3′)-III and showed high-level resistance to gentamycin and streptomycin, MSSA-21 possessed aph(3′)-III and exhibited low resistance to gentamycin, streptomycin, and kanamycin. The remaining three isolates (GRSA-2, GRSA-4, and GRSA-6) exhibited low resistance to all the aminoglycosides because they lack aminoglycoside-modifying enzyme coding genes in their genome. The transmission electron microscopy of the three isolates revealed changes in cell size, shape, and septa formation, supporting the view that the phenomenon of adaptive resistance is operative in these isolates.     

Overexpression of <Emphasis Type="Italic">AP1-</Emphasis>like genes from <Emphasis Type="Italic">Asteraceae</Emphasis> induces early-flowering in transgenic <Emphasis Type="Italic">Chrysanthemum</Emphasis> plants

Chrysanthemum is one of the most important commercial cut flowers in the world. Early-flowering cultivars are required to produce quality chrysanthemum flowers with a lower cost of production. To shorten the vegetative growth phase of chrysanthemum, three AP1-like genes from Asteraceae were constitutively overexpressed in 80 independent transgenic chrysanthemum lines. All lines were characterized by PCR and RT-PCR and demonstrated that overexpression of compositae AP1-homologs in transgenic chrysanthemum under long-day conditions had no effect on plant development compared to non-transgenic controls. Conversely, under short-day conditions, transgenic plants commenced bud initiation 2 wk earlier than non-transgenic chrysanthemum plants. Subsequently, transgenic chrysanthemum flowers showed color earlier and resulted in full opening of inflorescences 3 wk prior to non-transgenic control plants. These results open new possibilities for genetic improvement and breeding of chrysanthemum cultivars.     

Characterization of <Emphasis Type="Italic">EamaT1</Emphasis>, a member of <Emphasis Type="Italic">maT</Emphasis> family of transposable elements from the earthworm <Emphasis Type="Italic">Eisenia andrei</Emphasis> (Annelida,Oligochaeta)

The maT family is a unique clade within the Tc1-mariner superfamily, and their distribution is to date known as being limited to invertebrates. A novel transposon named EamaT1 is described from the genome of the earthworm Eisenia andrei. The full sized EamaT1 was obtained by degenerate and inverse PCR-based amplification. Sequence analysis of multiple copies of the EamaT1, which consisted of 0.9 and 1.4 kb elements, showed that the consensual EamaT1 with inverted terminal repeats (ITRs) of 69 bp was 1,422 bp long and flanked by a duplicated TA dinucleotide. The EamaT1 is present in approximately 120–250 copies per diploid genome but undergoes an inactivation process as a result of accumulating multiple mutations and is nonfunctional. The open reading frame (ORF) of the EamaT1 consensus encoding 356 amino acid sequences of transposase contained a DD37D signature and a conserved paired-like DNA binding motif for the transposition mechanism. The result of ITRs comparison confirmed their consensus terminal sequences (5′-CAGGGTG-3′) and AT-rich region on the internal bases for ITRs-transposase interaction.     

Infectivity analysis of two variable DNA B components of<Emphasis Type="Italic">Mungbean yellow mosaic virus-Vigna</Emphasis> in<Emphasis Type="Italic">Vigna mungo</Emphasis> and<Emphasis Type="Italic">Vigna radiata</Emphasis>

Mungbean yellow mosaic virus-Vigna (MYMV-Vig), aBegomovirus that causes yellow mosaic disease, was cloned from field-infected blackgram (Vigna mungo). One DNA A clone (KA30) and five different DNA B clones (KA21, KA22, KA27, KA28 and KA34) were obtained. The sequence identity in the 150-nt common region (CR) between DNA A and DNA B was highest (95%) for KA22 DNA B and lowest (85·6%) for KA27 DNA B. The Rep-binding domain had three complete 11 -nt (5’-TGTATCGGTGT-3′) iterons in KA22 DNA B (and KA21, KA28 and KA34), while the first iteron in KA27 DNA B (5’-ATCGGTGT-3’) had a 3-nt deletion. KA27 DNA B, which exhibited 93·9% CR sequence identity to the mungbean-infecting MYMV, also shared the 3-nt deletion in the first iteron besides having an 18-nt insertion between the third iteron and the conserved nonanucleotide. MYMV was found to be closely related to KA27 DNA B in amino acid sequence identity of BV1 (94·1%) and BC1 (97·6%) proteins and in the organization of nuclear localization signal (NLS), nuclear export signal (NES) and phosphorylation sites. Agroinoculation of blackgram (V. mungo) and mungbean (V. radiata) with partial dimers of KA27 and KA22 DNA Bs along with DNA A caused distinctly different symptoms. KA22 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in blackgram. In contrast, KA27 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in mungbean. Thus, DNA B of MYMV-Vig is an important determinant of host-range betweenV. mungo andV. radiata.     

In vitro propagation of two antidiabetic species known as guarumbo: <Emphasis Type="Italic">Cecropia obtusifolia</Emphasis> and <Emphasis Type="Italic">Cecropia peltata</Emphasis>

Two Cecropia species (Cecropia obtusifolia and C. peltata), known as guarumbo, are employed in Mexican traditional medicine to treat diabetes mellitus; the leaves of both species contain phenolic bioactive compounds such as chlorogenic acid (CA) and isoorientine (ISO), which have been attributed with hypoglycemic, hypolipidemic, and antioxidant properties. An in vitro propagation protocol was developed from existing apical bud meristem from C. obtusifolia seedlings; the shoot generation was induced on Murashige and Skoog (MS) medium supplemented with varying concentrations of 6-benzylaminopurine and kinetin (Kn) combined with either α-naphtalene acetic acid (NAA) or indole-3-acetic acid (IAA) auxins. Best morphogenetic response was developed with Kn 26.64 μM combined with either NAA or IAA 0.57 μM, respectively; likewise, C. peltata-seedling apical buds were subjected to these best selected treatments. Cecropia obtusifolia and C. peltata shoots were rooted in growth regulator-free half-strength MS medium, and regenerated whole plants were adapted successfully under greenhouse conditions and field. Leaves from both Cecropia-micropropagated plants produced the phenolic compounds CA and ISO, with highest concentrations in leaves from 18-month C. obtusifolia (12.28 ± 7.06 mg g⁻¹ dry leaves of CA and 8.30 ± 2.70 mg g⁻¹ dry leaves of ISO) growth in the field. Our results offer a protocol of apical-bud use for multiplication and curative-property conservation of the two previously mentioned important Mexican medicinal plants.