<Emphasis Type="Italic">CbLEA</Emphasis>, a Novel<Emphasis Type="Italic">LEA</Emphasis> Gene from<Emphasis Type="Italic">Chorispora bungeana</Emphasis>, Confers Cold Tolerance in Transgenic Tobacco

A novel late embryogenesis abundant (LEA) gene (AY804193), namedCbLEA, has now been isolated fromChorispora bungeana. This rare alpine subnival plant can survive sudden snowstorms and low temperatures. The full-lengthCbLEA is 842 bp, with an open reading frame encoding 169 ami no acids. The putative molecular weight ofCbLEA protein is 17.9 kDa, with an estimatedpl of 6.45. To investigate the functioning of thisCbLEA protein in cold-stress tolerance,CbLEA was introduced into tobacco under the control of the CaMV35S promoter. Second-generation (R₁) transgenic tobacco plants exhibited significantly increased tolerance to cold. These transgenics maintained lower malondialdehyde (MDA) contents and electrolyte leakage (EL) but their relative water content (RWC) was significantly higher compared with non-transgenic plants under chilling stress. Further experimental results showed that non-transgenic plants had severe freezing damage after exposure to -2°C for 1 h, whereas the transgenics suffered only slight injury under the same conditions. Moreover, survival was longer in the latter genotype at that temperature. The extent of increased cold tolerance was positive correlated with the level ofCbLEA protein accumulation, and was also reflected by the delayed development of damage symptoms. This indicates thatCbLEA is an excellent stress tolerance gene, and holds considerable potential as a new molecular tool for engineering improved plant genetics.     

High-frequency<Emphasis Type="Italic">in vitro</Emphasis> flowering in six species of<Emphasis Type="Italic">ceropegia</Emphasis>

High-frequencyin vitro flowering is reported here fromin vitro regenerated shoots ofin vitro-raised seedlings of rare and endemicCeropegia lawii, Ceropegia maccannii, Ceropegia oculata, andCeropegia sahyadrica, as well as the widely distributedCeropegia bulbosa var.bulbosa andCeropegia hirsuta. In our first set of experiments, the MS medium contained 87 mM sucrose and was supplemented with varying concentrations of BAP (4.4 to 26.6 μM). For the second set of trials, varying concentrations of sucrose (87 to 233 mM) were tested in MS media containing a constant 4.4 p.M BAP. Sub-cultured apical as well as axillary buds flowered with similar frequencies after 30 d of incubation. For all six species, the highest percentage of flowering shoots was obtained with either 26.6 μM BAP or 175 mM sucrose. Although smaller in size, theirin vitro flowers were morphologically comparable within wVo-derived flowers. Variations among species were noted for the number of flower buds per shoot and the percentage of flower formation. Because all six species showed similar responses in both experiments, we can suggest that this protocol is applicable across the wide range ofCeropegia species.     

Pollen morphology and its systematic implications for the genera<Emphasis Type="Italic">Keiskea</Emphasis> miq. and<Emphasis Type="Italic">Collinsonia</Emphasis> L. (Elsholtzieae-Lamiaceae)

The pollen morphology of six species ofKeiskea and three representative taxa ofCollinsonia was studied in detail using LM, SEM, and TEM. In both genera, pollen grains are monad, hexa-colpate, and mostly medium in size [P = 28.0 to 37.0 μm, E = 24.3 to 30.7 μm (Keiskea); P = 30.0 to 45.0 μm, E = 26.0 to 39.0 μm (Collinsonia)]. Polar outlines are of circular or ellipsoid form. Shapes range from primarily oblate-spheroidal to prolate-spheroidal to subprolate, and rarely prolate in the equatorial view. Their exine, including the inline characters, are clearly distinct from each other:Keiskea, well-developed bi-reticulate, often forming large lumina by supratectal ridges, unbranched columellae, one-third to one-half of the total exine thickness; versusCollinsonia, mostly perforate without supratectal ridges or a faint/very weak bi-reticulate appearance without supratectal ridges, seemingly branched columellae, ca. two-thirds of the total exine thickness. As demonstrated by these current data, the pollen morphology of the two genera is well distinguished, easily supporting the separation ofKeiskea fromCollinsonia.     

Mechanism by which<Emphasis Type="Italic">Bacillus</Emphasis>-Derived 2-Aminobenzoic acid inhibits the growth of<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Roots

To analyze the growth inhibitory mechanism of a 2-aminobenzoic acid (2-AA) derived fromBacillus cereus EJ-121, we treatedArabidopsis thaliana plants with 2-AA, 2-AA analogs, auxin (NAA), a known auxin transport inhibitor [2,3,5-triiodobenzoic acid (TIBA)], and an ethylene action inhibitor [silver thiosulfate (Ag)]. Root development was significantly inhibited by 50 μM 2-AA, whereas the growth of bacteria and yeast was undeterred. The application of two 2-AA analogs - 3-aminobenzoic acid (3-AA) and 4-aminobenzoic acid (4-AA) - did not impairArabidopsis root growth at concentrations below 100 μM. These results suggest that the effect of 2-AA is not due to its chemical structure, but because of its conversion to another metabolite, IAA. To confirm this, we supplemented TIBA in the growth medium, and found that the degree of inhibition was significantly reduced. Similarly, when plants were co-treated with 100 μM Ag, the negative effect of 50 μM 2-AA was greatly diminished. All of these observations support the proposal that this inhibition results from the conversion of 2-AA to IAA. Furthermore, the increased auxin level leads to a rise in ethylene synthesis, which then blocks root growth and, ultimately, retards overall plant development.     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of strictosidine using<Emphasis Type="Italic">lonicera japonica</Emphasis> leaf extracts and recombinant yeast

Strictosidine is a key intermediate in the biosynthesis of the terpenoid indole alkaloid (T1A) pathway. It results from a condensation reaction, catalyzed by strictosidine synthase (STR), between tryptamine and secologanin. We have now developed a useful method, based on enzyme-assisted synthesis, to produce strictosidine. Our procedure utilizes leaf extracts from Japanese honeysuckleLonicera japonica Thunb. as a secologanin source. In these experiments, an enzyme extract was prepared from transgenic yeastSaccharomyces cerevisiae that expresses theCatharanthus roseus STR (CrSTR) coding region. Strictosidine was then isolated with a 38% yield based on the initial amount of tryptamine in the enzymatic reaction.     

Peptide Fragments From Plant Vicilins Expressed in <Emphasis Type="Italic">Escherichia Coli</Emphasis> Exhibit Antimicrobial Activity <Emphasis Type="Italic">In Vitro</Emphasis>

Peptide fragments that exhibit antimicrobial activity in vitro have been shown to be produced by cleavage from the hydrophilic region near the N terminus of various vicilin proteins in plant seeds. Three peptide sequences identified in the hydrophilic region of vicilin seed proteins of Macadamia integrifolia and Theobroma cacao were predicted to exhibit antimicrobial activity based on sequence similarity to antimicrobial peptides that had been previously purified from macadamia kernels. Histidine-tagged versions of the putative antimicrobial peptides were expressed in Escherichia coli, purified, and demonstrated to have in vitro antimicrobial activity. There are many vicilin sequences in the growing plant genome sequence databases, and this expression method provides a high-throughput process for functionally testing the potential of internal peptide fragments of vicilins as novel antimicrobial molecules.     

Estimation of leaf number and leaf area of hydroponic pak-choi plants (<Emphasis Type="Italic">Brassica campestns</Emphasis> ssp,<Emphasis Type="Italic">chinensis</Emphasis>) using growing degree-days

Temperature is a principal environmental factor that directly affects the growth and timing of appearance for crop leaves. To estimate the leaf number and leaf area of ‘Seoul’ pak-choi plants (Brassica campestns ssp.chinensis), we applied the concept of growing degree-days GDD=(T_avg-T_base) × days, where T_avg, T_base and days were the daily average air temperature, base temperature, and days after transplanting, respectively. Leaves that were beginning to unfold with a leaf area ≥1 cm₂ were counted every 2 to 3 d. Linear relationships were found between leaf number and days after transplanting as well as between leaf number and GDD. The rate of appearance and the number of leaves per GDD were 0.542 leaves d^-1 and 0.051 leaves oC^-1 d^-1, respectively. In contrast, the relationship was non-linear between leaf number and leaf area, with the latter being calculated as [(128.9+11.6×GDD-0.03×GDD²)/1+(0.051×GDD+3.5) /13.7)^-3.9] (cm₂oC¹ d^-1). Using model validation, we found that the estimated leaf number and leaf area showed strong agreement with measured values. our results demonstrate the usefulness of modeling to estimate total leaf area and growth from hydroponically grown pak-choi plants.     

Taxonomic notes on<Emphasis Type="Italic">Linaria</Emphasis> mill. (<Emphasis Type="Italic">Scrophulariaceae</Emphasis>) for Flora Iberica

Taxonomic considerations and nomenclatural adjustments as a part of a revision ofLinaria Mill. undertaken within the “Flora iberica” project are presented. Data on morphology, seed coat surface, chorology, ecology, synonymy and variability of 11 accepted taxa are reported. The following new combinations are proposed:Linaria aeruginea subsp. cardonica (Font Quer)L. Sáez etM. Sainz,Linaria depauperata subsp.ilergabona (M.B. Crespo etV.J. Arán)L. Sáez, andLinaria saturejoides subsp.angustealata (Willmott)L. Sáez etM.B. Crespo.     

Co-expression of<Emphasis Type="Italic">flavonoid 3′ 5′-hydroxylase</Emphasis> and<Emphasis Type="Italic">flavonoid 3′-hydroxylase</Emphasis> Accelerates Decolorization in Transgenic Chrysanthemum Petals

Theflavonoid 3′,5′-hydroxylase (F3′,5′H) gene, derived from petunia, was introduced into chrysanthemum tissues by Agrobacterium-mediated genetic transformation. Cotyledon expiants were co-cultured withA. tumefaciens LBA 4404 harboring the vector pMBP that carriesF3′,5′H under the control of the CaMV 35S promoter andnptll as a selectable marker gene. After 72 h of co-cultivation, the expiants were placed on an MS medium supplemented with 4 mg L^-1 BA, 0.1 mg L^-1 NAA, 400 mg L^-1 carbenicillin, and 100 mg L^-1; kanamycin. After 4 weeks, kanamycin-resistant adventitious shoots had developed at a frequency of 6.3%. These shoots were then rooted and acclimatized in potting soil. Integration ofF3′,5′H into the plant genome was confirmed by Southern blot analysis. Flower buds that had red petals did not differ between the transgenic and the wild-type plants. However, petal color did change from red to bright orange to yellow when the buds developed into fully opened flowers on the transgenics. Spectrometric analysis revealed that the content of flavonoid compounds was more rapidly reduced in the transgenic petals as floral development proceeded. RT-PCR analysis showed thatF3′,5′H andflavonoid 3′hydroxylase (F3′H) were expressed simultaneously in the transgenic plants. Therefore, we suggest that this more rapid change in petal color results from 1) competition between levels of transgenicF3′,5′H and endogenousF3′H, each of which uses the same substrate in the flavonoid biosynthetic pathway and 2) the intrinsic substrate specificity of chrysanthemumDFR (dihydroflavonol 4-reductase).     

Effects of the aqueous extract from<Emphasis Type="Italic">artemisia campestris</Emphasis> ssp.<Emphasis Type="Italic">caudata</Emphasis> on mycorrhizal fungi colonization and growth of sand dune grasses

We used the aqueous extract fromArtemisia campesttis ssp.caudata to investigate its effects on the colonization of sand dune grass roots by mycorrhizal fungi and seedling growth. The percent colonization decreased with higher extract concentrations, and growth of three grass species was inhibited. Colonization by mycorrhizal fungi was more sensitive to the extract than was seedling growth, and no significant differences in the latter were found between the mycorrhizal and non-mycorrhizal treatments.     

Pollen and seed morphology of some<Emphasis Type="Italic">Ophrys</Emphasis> L. (Orchidaceae) taxa

Morphologies of pollen and seed surfaces were investigated with SEM technology for eight natural Ophrys taxa collected from southeastern Mediterranean countries and Turkey. The morphometric characteristics ofO. transhyrcana, O. sphegodes, O. epirotica, O. mammosa, O. pseudomammosa, O. oestrifera subsp.oestrifera, O. cornuta, andO. apifera were recorded and compared statistically. In general, the current species could be separated, in detail, according to those characteristics. These findings were also compared with those from recent studies, and were examined in terms of the present neighbouring taxa.     

Isolation of new microsatellite-containing sequences in<Emphasis Type="Italic">Acanthopanax senticosus</Emphasis>

Microsatellite markers, also called simple sequence repeats (SSRs), are comprised of a 2-to 6-nucIeotide repeat motif. They are useful as molecular markers for genetic authentication, crop breeding programs, and linkage analysis for map-based cloning. From a microsatellite-enriched genomic library ofAcanthopanax senticosus, we identified 239 new microsatellite-containing sequences. The di-nucleotide repeat units were the most abundant (55.2%), followed by tri-nucleotide repeat units (24.6%). In detailed repeat structures, the (AG)_n motif was most frequent (30.5%), followed by the (AC)_n motif (21.7%). Heptaand octa-nucleotide repeat motifs were found in each single locus, and a total of 33 (13.8%) complex repeat structures were recorded. This is the first report of mass isolation of microsatellites via screening of anA. senticosus library, and may well provide information useful as a genetic resource for the further study ofA. senticosus.     

Phylogenetic relationships in the genera<Emphasis Type="Italic"> Zostera</Emphasis> and<Emphasis Type="Italic"> Heterozostera</Emphasis> (Zosteraceae) based on<Emphasis Type="Italic"> matK</Emphasis> sequence data

Phylogenetic analysis of the plastid (chloroplast) DNA matK gene of Zosteraceae species was undertaken. A molecular phylogenetic tree based on matK sequence data showed the monophyly of Heterozostera tasmanica and subgenus Zosterella and did not support the separation of Heterozostera from the genus Zostera. The tree based on matK supported the monophyly of the subgenus Zostera, and showed that Zosteraceae consist of three main groups: Phyllospadix, which is clearly defined by being dioecious; the subgenus Zosterella and Heterozostera; and the subgenus Zostera. Character-state reconstruction of chromosome number and geographic distribution for our molecular phylogenetic tree showed that 2n=12 is a plesiomorphic character for Zostera and Heterozostera, that the chromosome number was doubled or tripled in two lineages, and that the initial speciation of Zostera and Heterozostera occurred in the Northern Hemisphere. The matK tree showed the close affinity of Z. noltii and Z. japonica, which have disjunct distributions. Zostera marina, which is the only widely distributed species in the subgenus Zostera, also occurring in the northern Atlantic, was shown to be embedded within other subgenus Zostera species.     

Association of <Emphasis Type="Italic">Bordetella</Emphasis> dermonecrotic toxin with the extracellular matrix

Background  

Bordetella dermonecrotic toxin (DNT) causes the turbinate atrophy in swine atrophic rhinitis, caused by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small amounts can affect target tissues is unknown.