Impact of pre-culture on short- and long-term in vitro recovery of the biosynthetic potential and enzymatic and non-enzymatic antioxidant defense of Hypericum rumeliacum Boiss. after cryostorage

Due to its high hypericin and pseudohypericin in vitro biosynthetic capacity, the Balkan endemic Hypericum rumeliacum was selected as a prospective candidate for long-term preservation of valuable medicinal plant germplasm. Initial cryopreservation experiments were previously conducted based on the successful protocol established and reported for the widely studied H. perforatum. This is the first report on the impact of pre-culture duration on the short- and long-term in vitro recovery of the biosynthetic potential and antioxidant defense system of H. rumeliacum cryopreserved by vitrification. Cryopreservation did not impair the phenolics and flavonoids production of the regenerated plants. Moreover, hypericin and pseudohypericin levels even increased substantially in one of the regenerated lines, reaching yields from 0.107 and 0.752?mg?g^?1?DW in the control up to 0.277 and 1.112?mg?g^?1?DW for hypericin and pseudohypericin, respectively. However, the physical injury stress of the pre-culture treatment manipulations affected the physiological status of regenerants in a time dependent manner. Within 6?months after thawing, regenerants with the highest oxidative stress after pre-culture, were characterized with an augmentation of antioxidant metabolites such as phenolics, flavonoids, glutathione and ascorbic acid as well as increased antioxidant enzymatic activities in comparison with both the non-frozen control and the regenerants with the lowest pre-culture oxidative stress. Then, after 18?months of recovery, the same first H. rumeliacum group displayed a marked drop of enzymatic antioxidant activity as compared with the other groups of plants. Further research is needed to target oxidative stress alleviation to optimize H. rumeliacum cryopreservation protocol.     

GenoFrag: software to design primers optimized for whole genome scanning by long-range PCR amplification

Genome sequence data can be used to analyze genome plasticity by whole genome PCR scanning. Small sized chromosomes can indeed be fully amplified by long-range PCR with a set of primers designed using a reference strain and applied to several other strains. Analysis of the resulting patterns can reveal the genome plasticity. To facilitate such analysis, we have developed GenoFrag, a software package for the design of primers optimized for whole genome scanning by long-range PCR. GenoFrag was developed for the analysis of Staphylococcus aureus genome plasticity by whole genome amplification in ~10 kb-long fragments. A set of primers was generated from the genome sequence of S.aureus N315, employed here as a reference strain. Two subsets of primers were successfully used to amplify two portions of the N315 chromosome. This experimental validation demonstrates that GenoFrag is a robust and reliable tool for primer design and that whole genome PCR scanning can be envisaged for the analysis of genome diversity in S.aureus, one of the major public health concerns worldwide.     

Monosomy 11Q: report of new phenotypic manifestations.

We present a case of new phenotypic findings not previously reported associated with a partial deletion of chromosome 11 with a break point at 23q - (46,XY,del(11)(q23). Partial deletion of chromosome 11q was first described by Jacobsen et al(4). Forty-eight patients have been reported during the last 30 years, with variable break points between 11q11 and 11qter. New phenotypic findings in our patient with the associated 11q deletion are imperforate anus, bilateral cataracts, and hypoplastic, multilobed lungs.     

EFFECTOR OF TRANSCRIPTION2 is involved in xylem differentiation and includes a functional DNA single strand cutting domain

EFFECTORS OF TRANSCRIPTION2 (ET) are plant-specific regulatory proteins characterized by the presence of two to five C-terminal DNA- and Zn-binding repeats, and a highly conserved cysteine pattern. We describe the structural characterization of the three member Arabidopsis thalianaET gene family and reveal some allelic sequence polymorphisms. A mutation analysis showed that AtET2 affects the expression of various KNAT genes involved in the maintenance of the undifferentiated state of cambial meristem cells. It also plays a role in the regulation of GA5 (gibberellin 3-beta-dioxygenase) and the cell-cycle-related GASA4. A correlation was established between AtET2 expression and the cellular differentiation state. AtET-GFP fusion proteins shuttle between the cytoplasm and nucleus, with the AtET2 product prevented from entering the nucleus in non-differentiating cells. Within the nucleus, AtET2 probably acts via a single strand cutting domain. A more general regulatory role for ET factors is proposed, governing cell differentiation in cambial meristems, a crucial process for the development of plant vascular tissues.     

Prompt response of superoxide dismutase and peroxidase to dehydration and rehydration of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The relic endemic nature of Haberlea rhodopensis, which grows in Balkan Peninsula, in combination with its high vegetative desiccation-tolerance, makes this species a good model to study mechanisms behind plant adaptation to severe drought stress. The aim of this study was to evaluate the antioxidant protection provided by Superoxide dismutase (SOD) and Peroxidase (PO) in H. rhodopensis after exposure to and recovery from dehydration at different developmental stages. During dehydration the electrolyte leakage from leaf tissue increased more significantly in post-flowering plants than in flowering plants, while upon subsequent rehydration this parameter showed a very fast decrease to the basic value of fresh leaves and did not depend on developmental stage. Like other higher plant species, SOD and PO demonstrated in H. rhodopensis an ability to adjust their activity very promptly to changing water supply. In addition, the leaves of this resurrection species retained significant activities of SOD and PO even in air-dried state, considered as the most severe form of water stress. The enhanced activity of antioxidant enzymes may either enable the scavenging of the active oxygen species produced at very severe water deficit, and/or carry a potential for resurrection on subsequent rehydration. Upon stress treatment total activities of both enzymes were higher in flowering than post-flowering plants which reveals that developmental stage might be a factor affecting plant stress tolerance. This work identified for the first time SOD isoforms of H. rhodopensis. Native PAGE showed at least six multiple isoforms in the protein extract from leaf tissue of flowering plants, and the differential visualization revealed that four of them were Cu, Zn-SOD isoforms, one was Mn-SOD and one Fe-SOD. These findings provide a good starting point for future study of the SOD gene family of this rare resurrection plant at the molecular level.     

Species richness and abundance of native leaf miners are affected by the presence of the invasive horse-chestnut leaf miner

The effect of the alien horse-chestnut leaf miner, Cameraria ohridella, on native fauna was studied by comparing the species richness of native leaf miner communities and the abundance of selected native leaf miner species in the presence and absence of horse-chestnut trees infested by C. ohridella, in various environments in Europe. The species richness of native leaf miner communities in Switzerland was lower at sites where C. ohridella was present than at control sites. In Switzerland, France and Bulgaria, several native leaf miner species were significantly less abundant in the vicinity of infested horse-chestnuts. The native species most affected by the presence of the invasive alien species were those occurring early in the year and sharing their parasitoid complex with C. ohridella. These results suggest apparent competition mediated by shared natural enemies because these are the only link between C. ohridella and native leaf miners using other food resources.     

Phytoplankton community of the drinking water supply reservoir Borovitsa (South Bulgaria) with an emphasis on cyanotoxins and water quality

The phytoplankton diversity, algal biomass, and selected physicochemical parameters were investigated in the drinking water reservoir (Borovitsa) located in the Kardzhali region, Bulgaria. Particular attention was given to Cyanoprokaryota and presence of cyanotoxins in the water samples. Twenty-nine species belonging to six divisions (Cyanoprokaryota, Chlorophyta, Zygnemophyta, Dinophyta, Euglenophyta and Bacillariophyta) were identified. The microscopic examination of the phytoplankton samples showed the dominance of Ankyra judayi, Oocystis lacustris (Chlorophyta) and Aphanizomenon flos-aquae (Cyanoprokaryota) in July 2006, and Microcystis pulverea, Synechococcus elongatus (Cyanoprokaryota), Radiococcus planktonicus (Chlorophyta) and Melosira varians (Bacillariophyta) in September 2006. A blooming event due to Aphanizomenon flos-aquae was observed in July 2006. The reservoir exhibits a tendency to shift from an oligotrophic environment to a state of mesotrophy. Presence of cyanotoxins such as anatoxin-a, microcystins and saxitoxins were analyzed by HPLC and ELISA methods. Our results demonstrated the presence of anatoxin-a and microcystins (0.09 μg/L) in the raw water samples from July 2006, and saxitoxins (2.5 μg/L) and microcystins (0.18 μg/L) in the raw water samples from September 2006. The study underlines that permanent monitoring programs of Cyanoprokaryota in the reservoirs used as sources of drinking water and toxicity assessments should be implemented. Indirect exposure and transfer of cyanotoxins through food chains must also be considered.     

SORTING NEXIN1 Is Required for Modulating the Trafficking and Stability of the Arabidopsis IRON-REGULATED TRANSPORTER1

Dicotyledonous plants growing under limited iron availability initiate a response resulting in the solubilization, reduction, and uptake of soil iron. The protein factors responsible for these steps are transmembrane proteins, suggesting that the intracellular trafficking machinery may be involved in iron acquisition. In search for components involved in the regulation of Arabidopsis thaliana iron deficiency responses, we identified the members of the SORTING NEXIN (SNX) protein family. SNX loss-of-function plants display enhanced susceptibility to iron deficiency in comparison to the wild type. The absence of SNX led to reduced iron import efficiency into the root. SNX1 showed partial colocalization with the principal root iron importer IRON-REGULATED TRANSPORTER1 (IRT1). In SNX loss-of-function plants, IRT1 protein levels were decreased compared with the wild type due to enhanced IRT1 degradation. This resulted in diminished amounts of the IRT1 protein at the plasma membrane. snx mutants exhibited enhanced iron deficiency responses compared with the wild type, presumably due to the lower iron uptake through IRT1. Our results reveal a role of SNX1 for the correct trafficking of IRT1 and, thus, for modulating the activity of the iron uptake machinery.