Functional hypothesis on miraculin’ sweetness by a molecular dynamics approach

Miraculin differs from other sweet-tasting proteins because it is a taste-modifier having the unusual property of modifying sourness into sweetness. Its dimer is covalently linked by an inter-chain disulphide bond, and shows its taste-modifying activity at acidic pH, with maximum at pH 3.0, while it is flat at neutral pH. Previous studies suggested the importance of two histidine residues for the taste-modifying activity of miraculin. In this work, we have conducted molecular dynamics simulations on wild type miraculin and on three mutated dimers (H29A, H59A and H29A/H59A) both at neutral and acidic pH to investigate the structural and functional role of these two His residues. Our results suggested that at acidic pH the presence of two charged His at the interface induced a structural rearrangement of the two monomers, thus leading to their relative opening and the following adaptation of their conformation to the receptor surface. On the other hand the simulations on three mutants showed that the mutated dimers had a closed form, and highlighted the important role of H29 in stabilizing/destabilizing the dimer arrangement and also a cooperative effect of the two histidines.     

Interactions between the human sweet-sensing T1R2-T1R3 receptor and sweeteners detected by saturation transfer difference NMR spectroscopy

The sweet receptor is a member of the G-protein coupled receptor family C that detects a wide variety of chemically and structurally diverse sweet-tasting molecules. We recently used saturation transfer difference spectroscopy (STD) to monitor the direct binding of a set of sweet agonists and antagonists to the human taste receptor in membranes prepared from human embryonic kidney (HEK293) cells transfected with and expressing the sweet receptor [F.M. Assadi-Porter, M. Tonelli, E. Maillet, K. Hallenga, O. Benard, M. Max, J.L. Markley, J. Am. Chem. Soc. 130 (2008) 7212-7213]. Here we review this work and related studies, discuss the procedures involved, and expand on their potential for identifying specific binding interactions of ligands to the membrane spanning and extracellular regions of the full heterodimeric sweet taste receptor. Whereas activity assays are unable to distinguish mutations that alter ligand-binding sites from those that alter signal transduction downstream of the binding site, STD NMR now allows us to make this distinction.     

Increased growth in elevated [CO2]: an early,short-term response?

Saplings of four clones of Sitka spruce and cherry were grown for three and two growing seasons, respectively, in open top chambers at two CO₂ concentrations (≈ 350 and ≈ 700 μmol mol^–1) to determine whether the increase in total biomass brought about by enhanced [CO₂] is a result of a transient or persistent effect in nonlimiting conditions. Classical growth analysis was applied to both species and mean current relative growth rate of total dry mass (R_T) and leaf dry mass (R_L), and period relative growth rate of total dry mass ( ) and leaf dry mass ( ) were calculated. Sitka spruce saplings and cherry seedlings showed a positive growth response to elevated [CO₂], and at the end of the experiments both species were ≈ 40% larger in elevated [CO₂] than in ambient [CO₂]. As a result, the period mean and were significantly higher in elevated [CO₂]. The differences in plant dry mass at the end of the experiments were a consequence of the more rapid growth in the early phase of exposure to elevated [CO₂]. After this initial phase mean R_T and R_L were similar or even lower in elevated [CO₂] than in ambient [CO₂]. NAR of both species was much higher in elevated [CO₂], whereas both LAR, SLA, and LMR showed the opposite trend. The higher LAR and SLA of plants in ambient [CO₂] contributed to a compensation by which they maintained R_T similar to that of elevated [CO₂] saplings despite lower NAR and photosynthetic rate. However, when the same size the trees were similar amongst the [CO₂] treatments, indicating that one of the main effect of elevated [CO₂] on tree growth is to speed-up early development in all aspects.     

Plant Regeneration and Transformation of Sweet Papper (Capsicum frutescens)

Three strains of sweet pepper, (Capsicum frutescens) “Shuang Feng”, “Zhong Jiao No. 2” and “Zhong Jiao No.3” were screened out of six Chinese cuhivars for their high capacity of regeneration. The normal flowering and fertile regenerated plants have been obtained from cotyledons of seedlings from 10 to 16 days old by a four-step culture procedure; short induction, shoot elongation, rooting of excised shoot and transplanting into soil. MS was the basal medium in all steps, supplemented with different kind and different concentration Of phytohomores. Optimal shoot ioduction medium is MS +4–6mg/LBA+0.5mg/LIAA which gives rise a shoot regeneration frequency of 100%. 35% of the induced shoots elongated on the medium of MS+2mg/L Zeatin or 2mg/L. BA+l–3mg/L GA, and subsequently rooted on MS medium or in addition of 0.1–0.5mg/LNAA. The regenerants were transplanted into soil and developed into normal plants. In the transformation of sweet pepper using the leaf disc method, two kinds of wild type Agrobacterium tumefaciens, C58 and GV3111, have been screened out in regard to their high infection capacity. The transient expression of GUS gene was detected and Kanamycin-resistant shoots from infected cotyledons have been obtained. Further assay and transfering the TMV-resistant and CMV-resistant genes into sweet pepper are in progress.     

Acclimatizing micropropagated black cherry by comparison with half-sib seedlings

Black cherry ( Prunus serotina Ehrh.) plantlets were cultured in vitro, transferred to potting mixture and evaluated for 8 weeks for acclimatization to a varying, but controlled environment. Whole plantlet growth and water relations were monitored and compared to seedlings of comparable size of the same maternal genotype grown under different conditions, but given the same pretest environment. At one week ex vitro. gravimetrically determined leaf conductance of plantlets was high, but became closer to that of seedlings in both magnitude and diurnal pattern as acclimatization progressed. By 8 weeks, leaf conductance of plantlets was nearly identical to that of seedlings, but the xylem water potential of the plantlets was significantly less. Logarithmic regressions of shoot vs root dry weight indicated that seedlings were allocating twice as much dry matter to shoot than to root growth compared to 4- and 8-week plantlets. Over the same period, stomatal densities of both seedlings and plantlets decreased and stomatal pore lengths increased. Multiple adventitious roots of plantlets emerged from a single site just below the root collar whereas secondary or lateral roots of seedlings originated acropetally along the central root axis. Leaf conductance of plantlets at 8 weeks was similar to that of seedlings indicating satisfactory acclimatization. The larger relative root growth rate of plantlets compared to the shoot, however, was associated with lower stem xylem water potential. The anomaly may be a consequence of the method of in vitro root formation. Plantlet growth rates were lower than growth rates of seedlings and their leaf area was correspondingly less.     

Identification and Genomic Variability of Prune dwarf virus Variants Infecting Stone Fruit Trees in Turkey

Prune dwarf virus (PDV), a pathogen of stone fruit trees worldwide, shows a great variability in its biological, serological and molecular properties. The coat protein sequences of ten PDV variants from different stone fruit tree species in Turkey were determined. The sequence for each variant was 657 nucleotides in length. The phylogenetic analyses of the sequences of the Turkish variants and of additional sequences of other PDV variants from international databases indicate the existence of four groups of PDV variants: one contains cherry variants that differs in group specific conserved amino acids; a second contains one apricot and cherry variants, most members of the group being Turkish variants; a third contains mixed variants from cherry, peach, plum trees and an almond tree; and a fourth contains only variants from almond trees. Based on their hosts, we propose the nomenclature of these groups as cherry I, cherry II, mixed and almond groups, respectively.     

甘薯兼抗薯瘟病和蔓割病种质筛选鉴定

用甘薯瘟2个致病型菌株对557份甘薯品种(系)分菌系做盆苗和田间接种鉴定,并用蔓割病菌接种鉴定.结果表明,筛选出兼抗薯瘟病2个菌系的品种(系)31份,兼抗甘薯瘟和蔓割病的品种(系)有16份,其中泉薯860、泉薯854、金山93、榕选416、金山908、广薯88-70和泉薯853等7份与我省主栽抗病品种湘薯75-55(CK)相比,具有类似的双抗性能力,而其抗瘟能力更强.福薯87、林泗2号、金山57和湛93-16等4份对照品种只抗单个病害或兼抗单个菌系.     

High Efficiency Organogenesis in Sweet Pepper (Capsicum annuum L.) Tissues from Different Seedling Explants

In vitro plant regeneration was achieved from eightsweet pepper varieties (Capsicum annuum L.). The effect ofvarious explant types (cotyledons, leaves, cotyledonary nodes and shoot-tip from25-day-old seedlings and embryonic cotyledons, embryonic hypocotyls and woundedseedlings) on bud and shoot regeneration and shoot elongation was evaluated.Differences in ability for in vitro shoot regeneration andelongation depended on the variety and explant type. In general, highregeneration frequency was obtained from all varieties. Agridulcedisplayed the highest regeneration response: an average of 3.45 elongated shootsper explant using embryonic cotyledons. Elongated shoots were excised and rootedon Murashige and Skoog (MS) basal medium either without plant growth regulatorsor with 0.5 IBA (indole-3-butyric acid) or 0.05 NAA (-naphthaleneacetic acid). Plantlets weretransplanted to soil and acclimatised in the greenhouse showing normaldevelopment and growing to maturity bearing normal fruits with seeds.