Identification of QTLs affecting adaptive traits in Castanea sativa Mill

A QTL analysis for three different adaptive traits was performed in an F₁ progeny of Castanea sativa Mill. The female and male parents originated from two Turkish chestnut populations adapted to a drought and humid environment, respectively. QTLs for bud flush, growth and carbon isotope discrimination were detected over a 3‐year period. Bud set was also recorded in the last year of measurement. Thirty‐five individual QTLs were detected for phenology, 28 for growth and 17 for carbon isotope discrimination, most of them explaining a low to moderate proportion of the total phenotypic variance. QTLs were distributed throughout the whole genome. Temporally stable QTLs were identified for all the traits analysed, with phenology showing the higher proportion of stable QTLs. Interesting phenotypic correlations and co‐localizations among QTLs for different adaptive traits were observed, allowing the formulation of an hypothesis about the genetic adaptation of the female parent to drought.     

Copper Excess Impairs Mobilization of Storage Proteins in Bean Cotyledons

Germination represents a limiting stage of plant life cycle. One of the underlying metabolic activities following imbibition of seed is the reserve mobilization. Seeds of bean (Phaseolus vulgaris L. var. soisson nain hatif) were germinated by soaking in distilled water or 200?μM CuCl(2). Storage proteins breakdown and amino acids freeing from reserve tissues were investigated. Compared to the control, Cu caused a reduction in germination rate, embryo growth, and in mobilization of cotyledonary biomass. The failure in albumin and globulin hydrolysis after the exposure to the pollutant was argued by (1) higher contents of remaining proteins than control ones, (2) persistence of some polypeptide bands resolved by polyacrylamide gel electrophoresis of albumin and globulin-rich fractions, and (3) decrease in the availability of amino acids. Nitrogen starvation in embryonic axis should be associated with the Cu-imposed delay in growth.     

Rooting Substances in Water Extracts of Castanea sativa and Salix viminalis

The rooting activity of water extracts of cuttings of chestnut (Castanea sativa Mill.) and willow (Salix viminalis L.) was studied throughout the year by the bean rooting test. The chestnut extract nullified the rooting activity of 10^?5M IAA, but on the whole it did not modify the rooting pattern of the bean cuttings. The willow extract increased rooting and showed a rhythm in the content of endogenous growth substances. IAA was found at the time when the extract showed the strongest rooting activity. The extract from willow contained two synergistic root-promoting fractions with the R_f's of 0.0–0.2 and 0.6–0.8 in isopropanol: ammonia: water. The latter one promoted rooting without added IAA. In this fraction a high amount of catechol was detected. No clear correlation was found between the ability to induce roots and the total phenol content in the two species.     

Earlywood vessels of Castanea sativa record temperature before their formation

The aim of this study was to identify the climatic signal contained in the earlywood vessel size of the ring-porous chestnut (Castanea sativa) and the physiological processes involved in the underlying mechanisms. In order to assign the encoded signal to a specific physiological process, bud phenology and vessel formation were monitored along an elevation transect and chronologies of the size of the first row of earlywood vessels were retrospectively correlated with 40 yr of early spring temperatures. The first vessels appeared in late April to early May, after encoding both a negative temperature signal in February-March (during tree quiescence) and a positive temperature signal in early April (at the time of resumption of shoot growth). We hypothesize that February and March temperatures affect cambial sensitivity to auxin, preconditioning tree responses later in the season. Furthermore, April temperature is related to tree activation whereby new hormone production fosters vessel expansion.     

Amino Acids Content in Germinating Seeds and Seedlings from Castanea sativa L

During germination the chestnut (Castanea sativa L.) var ecotype 33 accumulates a large amount of asparagine in the cotyledons. This compound also accumulates in the growing axis:shoots and roots. In the cotyledons, γ-aminobutyrate (GABA) represents a major amino compound during germination and early seedling growth. In young seedlings, 35 days old, arginine predominates over the other soluble amino acids, particularly in roots. Five enzymic activities involved in arginine and GABA have been measured in the storage organ of the seed: arginase and ornithine carbamyltransferase decrease during germination indicating the slowing down of the urea cycle. In contrast, ornithine aminotransferase increases. Glutamate decarboxylase is particularly active about 21 days after imbibition and GABA aminotransferase activity decreases during germination. These two activities are in good agreement with the likely transport of GABA from cotyledons to growing axis. Asparagine, arginine, and GABA are the three amino compounds obviously involved in the mobilization of nitrogen reserves in the germinating chestnut seeds Castanea sativa.     

Plant Proteins Are Smaller Because They Are Encoded by Fewer Exons than Animal Proteins

Protein size is an important biochemical feature since longer proteins can harbor more domains and therefore can display more biological functionalities than shorter proteins. We found remarkable differences in protein length, exon structure, and domain count among different phylo-genetic lineages. While eukaryotic proteins have an average size of 472 amino acid residues (aa), average protein sizes in plant genomes are smaller than those of animals and fungi. Proteins unique to plants are ?81 aa shorter than plant proteins conserved among other eukaryotic lineages. The smaller average size of plant proteins could neither be explained by endosymbiosis nor subcellular compartmentation nor exon size, but rather due to exon number. Metazoan proteins are encoded on average by ?10 exons of small size [?176 nucleotides (nt)]. Streptophyta have on average only ?5.7 exons of medium size (?230 nt). Multicellular species code for large proteins by increasing the exon number, while most unicellular organisms employ rather larger exons (>400 nt). Among sub-cellular compartments, membrane proteins are the largest (?520 aa), whereas the smallest proteins correspond to the gene ontology group of ribosome (?240 aa). Plant genes are encoded by half the number of exons and also contain fewer domains than animal proteins on average. Interestingly, endosymbiotic proteins that migrated to the plant nucleus became larger than their cyanobacterial orthologs. We thus conclude that plants have proteins larger than bacteria but smaller than animals or fungi. Compared to the average of eukaryotic species, plants have ?34%more but ?20%smal-ler proteins. This suggests that photosynthetic organisms are unique and deserve therefore special attention with regard to the evolutionary forces acting on their genomes and proteomes.     

Biology and monitoring of Dryocosmus kuriphilus on Castanea sativa in Southern Italy

• 1 The cynipid gall wasp Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) is an invasive wasp in Europe and is one of the major pests of chestnuts worldwide. An investigation of this cynipid was started in Campania, Southern Italy, in 2008, with the aim of providing guidelines for its sustainable control.
• 2 The number of larvae/gall increased in the second year of colonization relative to the first one but thereafter decreased in the third year of colonization, whereas the volume of the galls after the initial increase in the second year of colonization became stable. Adult emergence generally peaked between the middle of June and July.
• 3 The only reliable method for assessing the presence of the pest at its earliest stage was through dissecting the buds because oviposition scars on the buds tended to disappear during the winter.
• 4 Yellow traps were shown to be an effective method for assessing the emergence of cynipid adults and could potentially be used to time chemical applications in those cases in which this control method is feasible.
• 5 No correlation was recorded between the relative durations of the final stages (pupae, pharate adults and emergence).
• 6 Possible control strategies for this invasive pest are discussed.

     

Translatable mRNAs for Chloroplast-Targeted Proteins in Detached Radish Cotyledons during Senescence in Darkness

A protein-import system prepared with isolated chloroplastswas used to monitor changes in levels of mRNAs for chloroplast-targetedproteins during dark-induced leaf senescence. Biologically activechloroplasts were isolated from young (9-day-old) and aged (14-day-old)radish cotyledons. Poly(A)⁺-RNA was prepared from radish cotyledonsthat had been detached from seedlings and placed in darknessto accelerate senescence. The RNA was translated in a wheatgerm system, and the products were added to an import systemprepared with chloroplasts from young cotyledons. Electrophoreticanalysis of the imported proteins suggested that most chloroplast-targeted proteins decreased in abundance during dark treatmentof cotyledons. However, the relative abundance of 38 stromaland three thylakoid proteins increased transiently or continuouslyamong the products of translation of RNA isolated during thecourse of senescence. The efficiency of the uptake of precursorproteins by chloroplasts isolated from aged cotyledons was lowerthan that by chloroplasts from young tissue. The chloroplastsfrom aged cotyledons more efficiently imported at least onestromal protein and one thylakoid protein than chloroplastsfrom the young tissue. The relative abundance of these two proteinsincreased among the products of translation of RNA from senescingcotyledons when tested in the uptake system with chloroplastsfrom young cotyledons. These results suggest that some nucleargenes for chloroplast-targeted proteins are expressed in senescingcotyledons more efficiently than in young tissue, and that themachinery for import of proteins into chloroplasts changes duringaging of the tissue to allow more efficient import of certainproteins that may be responsible for the senescence of the chloroplasts. ¹Present address: Kihara Institute for Biological Research,Yokohama City University, Mutsukawa 3-122-20, Minami-ku, Yokohama,232 Japan     

Cotyledon storage proteins as markers of the genetic diversity in Castanea sativa Miller

This study has been to analyse the useful nut globulin proteins as a marker of the genetic diversity in Castanea sativa. The evaluated populations were highly polymorphic for the globulins, being detected up to 35 polymorphic bands with a wide distribution among all the evaluated populations. Taken together for populations from all the chestnut regions, about 39.3% of total allelic variation was distributed among the populations. The estimates of genetic similarity between populations were clearly associated with the collecting site. This method of analysis of the nut storage proteins (globulins) could be a useful tool for the evaluation of genetic diversity in this and other species of the Fagaceae.     

In vitro conservation of chestnut (Castanea sativa) by slow growth

Slow growth storage has been achieved for Castanea sativa (cv. ‘Montemarano’) shoot cultures over a duration of 48 mo at a temperature of 8°C, where 82% of explants survived and were able to resume normal growth after transfer to standard culture conditions at 23°C. The evaluation of the chlorophyll content of leaves also showed no differences between material stored for 48 mo and control material subcultured at 23°C. With a storage temperature of 4°C, the survival of shoots was significantly lower at approximately 56% after 12 mo, and no plants recovered after 24-mo storage. The presence of 6-benzyladenine 0.44 μM in the culture medium proved to be necessary for the recovery of healthy shoots, while pre-treatments with different concentrations of abscisic acid did not significantly influence the survival of shoots following storage conditions. A low level of light during slow growth storage resulted in positive effects on the rate of shoot survival over the longest preservation periods.     

Bacterial expression of an active class Ib chitinase from Castanea sativa cotyledons

Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.     

Microsatellite-based characterization of the Castanea sativa cultivar heritage of southern Switzerland.

Southern Switzerland has a long tradition of chestnut cultivation as a staple food. Local inhabitants constantly selected varieties according to the ripening period, the type of use, and the adaptability to the territory. As a result, the panorama of chestnut varieties is very complex, as reflected by more than 120 different variety names in an area of 26,000 ha. Since 1994, 47 varieties have been conserved in the chestnut germplasm of southern Switzerland (CSS), including Marroni, Euro-Japanese, and French varieties. A selection of 164 individuals from the CSS was analysed by 8 SSR markers (4 of which were developed in this study). Microsatellite analysis indicated that the CSS was accurately established, as 86% of the individuals grafted were correctly labeled. The identification of 98 genotypes, 10 clonal chestnut groups, 4 synonym groups, and 12 homonym groups reflected the complex ethnogeographical structure of the chestnut distribution. The 17 Marroni individuals considered clustered in 2 differentiated genetic groups instead of only 1 as expected. The fundamental problem of the frequent cases of homonymy and synonymy is discussed, as is the need for criteria for discriminating between polyclonal varieties and distinct homonymous varieties.     

Somatic Embryogenesis and Analysis of Peroxidase in Cultured Lettuce (Lactuca sativa L.) Cotyledons

Somatic embryos were induced in lettuce cotyledons culturedon Murashige and Skoog's (MS) medium containing either 2 mgl^–1 6-benzylaminopurine (BA) and 0.2 mg l^–1 naphthaleneaceticacid (NAA) or 0.2 mg l^–1 BA and 2 mg l^–1 NAA. Bothcombinations induced a frequency of over 70%. The explants culturedonly in the presence of 2,4-dichlorphenoxyacetic acid (2,4-D)did not produce somatic embryos. The development of the embryoidswas studied histologically and by scanning electron microscopy.Peroxidase activity was assayed and the isoenzyme pattern ofcalluses was determined by polyacrylamide gel electrophoresis.Callus from an embryogenic line showed a much higher peroxidaseactivity than that from a non-embryogenic line, one extra peroxidaseisozyme band being present and typical of the embryogenic callus.No qualitative differences were detectable between the embryogeniccalluses. Lactuca sativa L, lettuce, somatic embryogenesis, peroxidases, isoenzymes     

Are Odorant-binding Proteins Involved in Odorant Discrimination?

Pheromone-sensitive sensilla trichodea of nine moth speciesbelonging to six families and three superfamilies of Lepidopterawere immunolabelled with an antiserum against the pheromone-bindingprotein of Antheraea polyphemus. Strong immunolabelling of thesensillum lymph was observed in all long sensilla trichodeaof A. polyphemus, A. pernyi (Saturniidae), Bombyx mori (Bombycidae)and Manduca sexta (Sphingidae). Very weak labelling was foundwith all sensilla trichodea of Dendrolimus kikuchii (Lasiocampidae)and Lymantria dispar (Lymantriidae). In three noctuid species,some long sensilla trichodea were labelled strongly, some onlyweakly and some were not labelled at all. The fraction of longsensilla trichodea that were strongly labelled was large inHelicoverpa armigera, but small in Spodoptera littoralis andAutographa gamma. The observed cross-reactivity was not correlatedwith taxonomic relatedness of the species but rather with chemicalrelatedness of the pheromones used by these species, as a highlabelling density was consistently observed in sensilla tunedto pheromones with an alcyl chain of 16 carbon atoms. The highlydivergent specificity of pheromone-receptor cells in Noctuidaeappears to be mirrored by a similar diversity of the pheromone-bindingproteins in the sensilla trichodea. These data support the notionthat pheromone-binding proteins participate in odorant discrimination.Chem. Senses 21: 719–727, 1996.     

Utilization of Response Surface Methodology in Optimization of Proline Extraction from Castanea sativa Honey

Proline constitutes approximately 85 % of the amino acid composition of honey. Therefore, the quantitative determination of this amino acid in honey samples is used by many national/international authorities to evaluate the quality of honey types. In this study, it was aimed to achieve maximum proline amino acid extraction from honey samples whose botanical origins were confirmed by melissopalynological analysis. For this reason, based on three different spectrophotometric methods used in the literature for proline analysis, proline extraction was optimized with the Response Surface Method (RSM) and Box-Behnken experimental design. Three independent variables were determined as treatment time (2, 6, and 10 min), treatment temperature (22, 46, and 70 °C), and cooling time (5, 25, and 45 min). As a result of the optimization, it was seen that only significantly effective independent variable on the proline content of honey was the processing temperature. The optimum conditions obtained as a result of the RSM were found to be 2 min for the treatment time, 70 °C for the treatment temperature and 45 min for the cooling time. The composite desirability of the optimum conditions (R²) was found to be 1.00. It was determined that the method proposed by International Honey Commission (IHC) is efficient for proline analysis, but it provides more proline extraction by reducing of time from 10 min to 2 min in hold time in boiling water bath only during the extraction step. As a result, the conditions to be used in order to achieve maximum proline extraction with different spectrophotometric methods were determined and optimum values were determined. In addition, since the botanical origin of honey samples significantly affects the proline content of honey, it can be suggested that this study be optimized for different monofloral honey samples as well.     

Isolation and characterization of polymorphic microsatellites in European chestnut (Castanea sativa Mill.)

Thirteen polymorphic microsatellite loci were isolated from Castanea sativa (Mill.). Six contained dinucleotide repeats, six contained trinucleotide repeats, and one contained a compound microsatellite of a trinucleotide and a tetranucleotide repeat. The loci were characterized using C. sativa trees from three populations in the UK and the parents and six seedlings from a Turkish mapping population. The number of alleles revealed varied from two to 14 (mean = 5.15) per loci. Eight loci were found to be useful in the mapping family.