An approach towards genetically engineered cell fate mapping in maize using the Lc gene as a visible marker: transactivation capacity of Lc vectors in differentiated maize cells and microinjection of Lc vectors into somatic embryos and shoot apical meristems

To establish a system for genetically engineered cell fate mapping, different vectors carrying the Lc gene, a member of the R gene family, were delivered into embryonic and meristematic cells of maize by the microinjection technique. Vectors in which the Lc cDNA is driven either by a constitutive promoter (CaMV 35S), with or without the Adh1 intron 1 of maize, or a tissue-specific promoter (phosphoenolpyruvate carboxylase, PEPC) as well as self-replicating wheat dwarf virus (WDV) vectors carrying a Lc-expression-cassette, have been tested. The ability of these vectors to transactivate was evaluated in mesophyll-derived protoplasts of the maize genotype appropriate for these microinjection experiments. The expression product of the introduced Lc gene can substitute for mutated R and B loci, resulting in anthocyanin production. Analogous results were obtained by microinjection into organized tissues, where transactivation of anthocyanin biosynthesis resulted in pigmented sectors in somatic embryos (B79) and in the leaves of plants regenerated from the cultivated shoot apical meristems (K55, r-g, b). The tissue-specific appearance of pigmented sectors in leaves, using the mesophyll-specific PEPC promoter suggests the possibility of using this approach for layer-specific cell fate studies. The presence of the introduced plasmids in leaves showing red sectors 20–30 days after injection was proven by PCR analysis.     

Anthocyanin accumulation enhanced in <Emphasis Type="Italic">Lc</Emphasis>-transgenic cotton under light and increased resistance to bollworm

Breeding of naturally colored cotton fiber has been hampered by the limited germplasm, an alternative way is to use transgenic approach to create more germplasm for breeding. Here, we report our effort to engineer anthocyanin production in cotton. The maize Lc gene, under the control of the constitutive 35S promoter, was introduced into cotton through genetic transformation. Our data showed that the expression of the Lc gene alone is sufficient to trigger the accumulation of anthocyanin in a variety of cell types including fiber cells in cotton. However, the accumulation of colored anthocyanin in cotton fibers requires the participation of light signaling. These data indicate that it is feasible to engineer colored fibers through transgenic approach in cotton. Furthermore, we showed that the Lc-transgenic cotton plants are resistant to cotton bollworm. These transgenic plants are, therefore, potentially useful for cotton breeding against cotton bollworm.     

Death and survival of heterozygous Lurcher Purkinje cells In vitro

The differentiation and survival of heterozygous Lurcher (+/Lc) Purkinje cells in vitro was examined as a model system for studying how chronic ionic stress affects neuronal differentiation and survival. The Lurcher mutation in the δ2 glutamate receptor (GluRδ2) converts an orphan receptor into a membrane channel that constitutively passes an inward cation current. In the GluRδ2^+/Lc mutant, Purkinje cell dendritic differentiation is disrupted and the cells degenerate following the first week of postnatal development. To determine if the GluRδ2^+/Lc Purkinje cell phenotype is recapitulated in vitro, +/+, and +/Lc Purkinje cells from postnatal Day 0 pups were grown in either isolated cell or cerebellar slice cultures. GluRδ2^+/+ and GluRδ2^+/Lc Purkinje cells appeared to develop normally through the first 7 days in vitro (DIV), but by 11 DIV GluRδ2^+/Lc Purkinje cells exhibited a significantly higher cation leak current. By 14 DIV, GluRδ2^+/Lc Purkinje cell dendrites were stunted and the number of surviving GluRδ2^+/Lc Purkinje cells was reduced by 75% compared to controls. However, treatment of +/Lc cerebellar cultures with 1‐naphthyl acetyl spermine increased +/Lc Purkinje cell survival to wild type levels. These results support the conclusion that the Lurcher mutation in GluRδ2 induces cell autonomous defects in differentiation and survival. The establishment of a tissue culture system for studying cell injury and death mechanisms in a relatively simple system like GluRδ2^+/Lc Purkinje cells will provide a valuable model for studying how the induction of a chronic inward cation current in a single cell type affects neuronal differentiation and survival. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Displaced and tandem duplications in the long arm of chromosome 10 in maize

Lc, an anthocyanin pigmenting factor mapping somewhat more than one unit distal to R, is borne on a chromosomal segment which is homologous with part of the R-r:standard duplicated segment. Deficiencies and tandem duplications of the R to Lc region arise from exchanges within these obliquely paired homologous segments. The deficiencies are transmitted with a high, although reduced, frequency by the male gametophyte and are homozygous viable. Yet, the R to Lc region is not duplicated either proximal to R or distal to Lc. Thus the Lc-marked segment and either the P- or the S-marked segment of R-r constitute a displaced duplication. Such an arrangement can initiate a tandem and displaced duplication cycle.———No evidence was obtained for fractionation of the compound phenotype conditioned by Lc.     

TREGED: A new strategy for inducing deletions in plant genomes

We have created a DNA construct, TREGED (transposon-and recombinase-mediated genome deletion), that will automatically induce deletions in plant genomes. TREGED contains the maizeAc/Ds transposon, the yeast R-RS site-specific recombination system, the bacterialtetR repression systems, a novel artificial superintron, and the marker genesGUS andLc. The novelty of TREGED is that only one cross is required to trigger a sequence of events leading to deletion and, simultaneously, to a color assay to detect the deletion. Crossing is done to introduceAc transposase which activatesDs transposition from TREGED to a nearby chromosome region.Ds transposition, in turn, activates recombination between an engineeredRS site on TREGED and anRS site on the transposedDs fragment, thus deleting the genome segment between TREGED andDs. The recombination event also deletesLc orGUS and part oftetR, which triggers expression ofGUS orLc color genes for an upstream or downstream deletion respectively. Each TREGED insertion site will produce multiple kinds of deletions identifiable by inspecting a single F₁ plant and its progeny for colored tissue. The color markers can also be used to differentiate between deletion and other more rare events such as translocation and inversion. We anticipate TREGED will greatly simplify the steps required to obtain useful deletions—eventually allowing the creation of detailed deletion libraries. Such libraries will be particularly useful for anlaysis of gene and chromatin function in plant species with large genomes.     

Medium optimization for cell biomass yield and anthocyanin production in cell suspension culture of Melastoma malabathricum

Macronutrients influence the cell biomass yield and anthocyanin production in plant cell suspension cultures. However, different species respond differently for different nutrient concentrations. In this study, we tested the effect of different concentrations of macronutrients on the cell cultures of Melastoma malabathricum L. The results showed that the addition of NH₄NO₃ did not show any increment in the cell biomass but it enhanced the anthocyanin accumulation. Results indicated that CaCl₂.2H₂O and MgSO₄.7H₂O played a more important role in anthocyanin accumulation than the cell growth of M. malabathricum. However, the presence or absence of KNO₃ and KH₂PO₄ did not show any effect on either the cell biomass or the anthocyanin production.     

A new allele of the lurcher gene, lurcher

A new neurological mouse mutation that arose spontaneously in a BALB/cByJ stock displays a semidominant pattern of inheritance. In the heterozygote, this mutation results in an early loss of Purkinje cells in the cerebellum, which is followed by the overt symptom of an ataxic gait first observed at postnatal day 13 (P13). A portion of animals homozygous for the mutation die within P0; the remaining homozygotes die by P25. The mutation maps to mouse Chromosome (Chr) 6 between markers D6Rck314 and D6Rck361, a chromosomal segment that contains the lurcher (Lc) locus. The Lc mutation is also semidominant and has a strikingly similar phenotype. A cross between a new mutant (Nm) heterozygote and an Lc heterozygote yields double heterozygotes, animals that carry both mutations, with a phenotype similar to that of both Nm and Lc homozygotes. The similarity in phenotype, the colocalization of the two loci on mouse Chr 6, and the positive result of the allelism test demonstrate that the new mutation is an allele of the Lc gene. Received: 4 April 1997 / Accepted: 21 April 1997     

β-Defensin from the Asian Sea Bass,Lates calcarifer: Molecular Prediction and Phylogenetic Analysis

Antimicrobial peptides (AMPs) are an important element of the innate immune system of all living organisms and serve as a barrier that safeguards the organisms against a wide range of pathogens. Fishes are proven to be a prospective source of AMPs, and β-defensins form an important family of AMPs with potent antimicrobial, chemotactic and immunomodulatory activities. The present study reports a β-defensin AMP sequence (Lc-BD) from the Asian sea bass, Lates calcarifer, a commercially important fish species in tropical and subtropical regions of Asia and the Pacific. A 202-bp cDNA fragment with an open reading frame encoding 63 amino acids (aa) was obtained from the mRNA of gill tissue by RT-PCR. The deduced aa sequence of Lc-BD possessed a signal and a mature peptide region with 20 and 43 aa residues, respectively. Lc-BD was characterized at the molecular level, and a molecular weight of 5.24 kDa and a net charge of +4.5 was predicted for the mature peptide. The molecular characterization of Lc-BD revealed the presence of three intramolecular disulphide bonds involving the six conserved cysteine residues in the sequence, and the phylogenetic analysis of Lc-BD showed a close relationship with β-defensins from fishes like Siniperca chuatsi, Argyrosomus regius, Trachinotus ovatus and Oplegnathus fasciatus.

     

Overexpression of a Hu-bcl-2 transgene in Lurcher mutant mice delays Purkinje cell death

Cerebellar Purkinje cells in the heterozygous Lurcher mutant undergo cell autonomous degeneration beginning in the second week of postnatal development and becoming almost total around 30–45 days. The Lurcher mutation was recently identified as gain-of-function defect in the δ2 glutamate receptor causing a constitutive current leak, suggesting that +/Lc Purkinje cells die by an excitotoxic mechanism. In previous studies we have shown that overexpression of bcl-2, a key regulator of cell death, in the heterozygous Lurcher mutant does not prevent +/Lc Purkinje cell death. To investigate further the mechanisms of +/Lc Purkinje cell death, we have crossed +/Lc mutants with a second line of Hu-bcl-2 transgenics (NSE73a) that shows an earlier onset of transgene expression and higher expression levels. Analysis of eight +/Lc-NSE73a mutants (4 at 2 months and 4 at 5–6 months) showed that Hu-bcl-2 overexpression delayed, but ultimately could not prevent +/Lc Purkinje cell death.     

Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

The effects of yeast extract and selected polysaccharide elicitors on secondary metabolite production, particularly of anthocyanin and phenolic acid, in cell suspension cultures of Vitis vinifera were investigated. All elicitors either maintained or promoted cell growth in culture. Overall, secondary metabolite production in V. vinifera cell suspension cultures responded differently to different elicitors. Chitosan, pectin, and alginate enhanced production of anthocyanin within 13 days of culture with levels of 2.5-, 2.5-, and 2.6-fold increase, respectively, over that of control. Chitosan, alginate, and gum arabic significantly promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in V. vinifera cultures, as well as in the culture medium. Intracellular phenolic acid production was significantly enhanced by alginate and chitosan, with 1.7- and 1.5-fold levels, respectively, of that of control. Extracellular phenolic acid production was also significantly increased in the presence of chitosan and gum arabic, with levels of 3.3- and 1.7-fold higher, respectively, than those of control. In addition, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.     

Multiple Bacteriocin Production by Leuconostoc mesenteroidesTA33a and Other Leuconostoc/Weissella Strains

Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins. Received: 11 April 1997 / Accepted: 10 June 1997     

A combination of elicitation and precursor feeding leads to increased anthocyanin synthesis in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Both elicitation and precursor feeding are effective strategies for improving secondary metabolite production in plant cell suspension cultures. In this study, cell suspension cultures of Vitis vinifera subjected to methyl jasmonate treatment resulted in a significant increase in levels of anthocyanin production. Moreover, a combination of 5 mg/L phenylalanine and 50 mg/L methyl jasmonate promoted the highest level of anthocyanin biosynthesis, resulting in 4.6- and 3.4-fold increases in anthocyanin content and yield, respectively, over the control. The optimum period for elicitation of anthocyanin synthesis was 4 days following incubation in the presence of elicitors, at the beginning of the exponential growth phase. V. vinifera cell lines of different anthocyanin-producing capabilities responded differently to elicitation and precursor feeding. Anthocyanin production of a low-producing cell line, VV06, could be enhanced with addition of elicitors and precursor feeding. Methyl jasmonate was the only elicitor that increased anthocyanin production of the high-producing cell line VV05, but contributed to moderate enhancement of anthocyanin production compared with VV06. For cell line VV06, synergistic effects were observed for all treatment combinations of methyl jasmonate along with other elicitors and precursors. In addition, 6.1- and 4.6-fold increases in anthocyanin content and yield, respectively, were obtained in the presence of 5 mg/L phenylalanine, 50 mg/L methyl jasmonate, and 1 mg/L dextran. However, none of these treatment combinations exhibited synergistic effects in cell line VV05.     

“HAIRY CANOLA” – Arabidopsis GL3 Induces a Dense Covering of Trichomes on Brassica napus Seedlings

Transformation with the Arabidopsis bHLH gene 35S:GLABRA3 (GL3) produced novel B. napus plants with an extremely dense coverage of trichomes on seedling tissues (stems and young leaves). In contrast, trichomes were strongly induced in seedling stems and moderately induced in leaves of a hairy, purple phenotype transformed with a 2.2 kb allele of the maize anthocyanin regulator LEAF COLOUR (Lc), but only weakly induced by BOOSTER (B-Peru), the maize Lc 2.4 kb allele, or the Arabidopsis trichome MYB gene GLABRA1 (GL1). B. napus plants containing only the GL3 transgene had a greater proportion of trichomes on the adaxial leaf surface, whereas all other plant types had a greater proportion on the abaxial surface. Progeny of crosses between GL3⁺ and GL1⁺ plants resulted in trichome densities intermediate between a single-insertion GL3⁺ plant and a double-insertion GL3⁺ plant. None of the transformations stimulated trichomes on Brassica cotyledons or on non-seedling tissues. A small portion of bHLH gene-induced trichomes had a swollen terminal structure. The results suggest that trichome development in B. napus may be regulated differently from Arabidopsis. They also imply that insertion of GL3 into Brassica species under a tissue-specific promoter has strong potential for developing insect-resistant crop plants. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.