Seasonal regulation of a 24-kDa protein from red-osier dogwood (Cornus sericea) xylem

Previously, we showed that an apparent cell wall-plasma membrane interaction in xylem ray parenchyma differed between cold acclimated and non-acclimated red-osier dogwood (Cornus sericea L.) (Ristic and Ashworth 1994). For the present study, a calcium chloride extraction method was used to identify cell-wall-associated xylem proteins that accumulated during periods of cold acclimation. A 24-kDa protein represented the predominant protein in both total protein and CaCl2 extracts during cold acclimation of field-grown plants. Two-dimensional gel electrophoresis separated the 24-kDa protein into four basic isoforms. The most abundant and basic isoform had a high glycine content. In-gel digestion of this basic 24-kDa isoform generated three partial peptide fragments, of which one exhibited homology to the dehydrin protein family. An anti-dehydrin polyclonal antibody cross-reacted with the 24-kDa protein, providing further evidence that this protein is related to dehydrins. The 24-kDa protein began to accumulate in late August, reached a maximum in midwinter, declined during the spring months and was absent in early summer.     

A molecular marker associated with low-temperature induction of dormancy in red osier dogwood (Cornus sericea)

Dormancy induction in temperate deciduous plants is thought to be regulated by short photoperiods, but low temperature has been shown to eliminate the short photoperiod requirement in northern ecotypes. An F2 population (191 plants) red osier dogwood (Cornus sericea L.) derived from a polycross of an F1 population produced from reciprocal crosses of the parental clonal ecotypes, Northwest Territories (NWT, 62 degrees N) and Utah (42 degrees N), was examined to identify molecular markers of temperature-induced endodormancy. Dormancy induction curves were generated for each individual in the F2 population and a standard point prior to vegetative maturity (i-VM) was inferred from the change in slope of the dormancy acquisition curve. Under Saskatoon, Saskatchewan field conditions (52 degrees N), the NWT ecotype entered i-VM on average 5-6 weeks before the Utah ecotype. Two sub-populations of the F2 population were distinguishable based on VM acquisition on exposure to low temperature but not to short photoperiods. A sequence characterized amplified region (SCAR) marker was developed that correctly (> 92%) identified individual plants within the F2 subpopulation that were responsive to low-temperature induction of VM. Timing of bud break was strongly associated with the timing of VM in the geographical ecotypes but not in the F2 population, indicating that these are separate traits under genetic control.     

Seasonal variations in soluble sugars and starch within woody stems of Cornus sericea L

Carbohydrate composition changed seasonally in red osier dogwood (Cornus sericea L.) stem tissues. Starch concentration was highest in fall and decreased to a minimum in midwinter. Coincident with the breakdown of starch in fall, there was an increase in the concentrations of soluble sugars. Soluble sugars were present in highest concentrations in midwinter. Glucose, fructose, sucrose, and raffinose were the predominant soluble sugars present in both bark and wood tissues. In early spring, the soluble sugar concentration decreased and the concentration of starch increased. The seasonal interchange between sugars and starch did not simply reflect a general quantitative shift in the balance between sugars and starch because qualitative changes in soluble sugars were also noted. The most striking changes involved the trisaccharide raffinose. Raffinose was barely detectable in summer and early fall, but increased to one fifth and one third of the total soluble sugars in January samples of bark and wood tissues, respectively. The potential physiological role of raffinose in overwintering red osier dogwood tissue is discussed.     

AFP抗冻蛋白基因工程研究进展

抗冻基因工程一直是生物技术领域研究的热点之一，本文综述了近年来国内外有关AFP抗冻蛋白基因工程的研究方向，进展及成就，并提出了此领域尚存在的一些问题及前景展望。     

The effects of canopy gaps on seedling emergence and growth of two deciduous broad-leaved species (Cornus controversa andPrunus grayana) in aCryptomeria japonica plantations

The effects of canopy gaps on seedling emergence and growth ofCornus controversa andPrunus grayana were studied in a 21-year-oldCryptomeria japonica plantation. The seeds of the two species were sown in December 1995 and their fate was followed until March 2000.P. grayana germinated in 1996, butC. controversa germinated in 1997. In both species studied, more than 70% of the seedlings survived in the forest edge until the end of the experiment, although none survived in the forest understory in the first growing season. In the gap, the survival rate was higher inP. grayana than inC. controversa. In this experiment, some trees were cut to enlarge the area of the gap, in which the growth rate increased markedly forC. controversa seedlings, but not forP. grayana seedlings after the cutting. These two species showed substantial differences in the patterns of seedling emergence, survival and growth in aCr. japonica plantation which had a canopy gap.     

Analysis of bark proteins in blister rust-resistant and susceptible western white pine (Pinus monticola)

We compared bark proteins from four contrasting (blister rust-resistant versus susceptible) half-sib seedling pairs of western white pine (Pinus monticola D. Don). Pooled proteins from resistant and susceptible groups (four trees per group) were separated by two-dimensional gel electrophoresis, silver stained, and analyzed with the aid of a laser scanner interfaced with a computerized gel documentation system. Qualitative and quantitative protein differences were observed between resistant and susceptible groups. The number of proteins unique to a group was greater in the susceptible category than in the resistant category. Biosynthesis of some common proteins was enhanced near lesioned areas of susceptible seedlings. Many proteins shared similar charge and mass characteristics with those of pathogenesis-related (PR) proteins. Two protein bands were isolated and partially characterized by N-terminal amino acid sequencing: a 10.6-kDa band that was selectively enriched in all resistant individuals, and a 26.0-kDa band that was enriched in some susceptible individuals. The significance of these protein differences and the possible use of selected proteins as disease or resistance markers are discussed.     

Analysis of needle proteins and N-terminal amino acid sequences of two photosystem II proteins of western white pine (Pinus monticola D. Don)

A method is described for extracting needle proteins from mature western white pine (Pinus monticola D. Don) trees. Extracted proteins were separated into 26 components by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The predominant protein component (32-39%) had a molecular weight of 57 kDa. The second most predominant protein component (14-25%) had a molecular weight of 16 kDa. The amino acid composition of the twenty-six protein components was determined and a few selected proteins were subjected to further amino acid sequence analysis. Of these, two proteins, designated as Pin m I and Pin m II, were identified by sequence homology with other Photosystem II proteins. The Pin m I and Pin m II proteins had molecular weights of 22.6 kDa and 23.4 kDa, respectively. Pin m I had a much higher proline content than Pin m II.     

Allometry, biomass, and chemical content of Novel African Tulip Tree (Spathodea campanulata) Forests in Puerto Rico

The African tulip tree, Spathodea campanulata, the most common tree in Puerto Rico, forms novel forest types with mixtures of native and other introduced tree species. Novel forests increase in area in response to human activity and there is no information about their biomass accumulation and nutrient cycling. We established allometric relationships and chemically analyzed plant parts of African tulip trees to determine the concentration and standing stock of chemical elements (C, N, P, K, Ca, Mg, S, Mn, Al, Fe, Na), and ash. Trees ranged in diameter at breast height from 8 to 85 cm and in height from 8.8 to 28 m. The concentrations of N, P, K, and Ca in leaves of the African tulip tree were similar to those of the native pioneer Cecropia schreberiana and higher than those of mature forest tree species in Puerto Rico. The over bark wood volume of African tulip trees in nine forest stands where it was dominant ranged from 163 to 849 m³/ha. Aboveground biomass ranged from 60 to 296 Mg/ha, and N and P stocks ranged from 190 to 988 and 32 to 137 kg/ha, respectively. Novel forests on abandoned agricultural lands can store more biomass and elements than native and plantation forest stands of similar age.     

Distribution and partial characterization of seasonally expressed proteins in different aged shoots and roots of 'Loring' peach (Prunus persica)

During autumnal leaf senescence, leaf nitrogen in deciduous trees is translocated to storage sites, especially bark and xylem tissues. Proteins that accumulate in large amounts in bark and xylem in winter and are absent from these organs in summer are called storage proteins, and are believed to be vehicles for storing nitrogen reserves. These reserves are important for spring growth and help trees tolerate or recover from both abiotic and biotic stresses. Based on seasonal patterns of accumulation, we previously identified three storage proteins with molecular masses of 60, 19 and 16 kDa in bark tissues of 'Loring' peach (Prunus persica (L.) Batsch). To characterize the distribution of these proteins in different-aged tissues and to determine if they have any function other than nitrogen storage, we examined their seasonal distribution in bark tissues of current-year and 1-year-old shoots, scaffold branches, main trunks and 4-5-year-old roots of 'Loring' peach. Verification of protein identity was based on molecular mass and reactions with antibodies directed against each specific protein. Protein distribution was variable. For all three proteins, the greatest amount was present in mid-winter in current-year and 1-year-old shoots. These tissues also showed the greatest seasonal variation in the amount of protein present. The 16 kDa protein was present only in the youngest shoots, whereas the 19 kDa protein was present in all tissues examined. The 60 kDa protein was absent in root tissue. The amino acid composition and sequence of each protein were determined. The 60 kDa protein was identified as a dehydrin, and the 19 kDa protein appeared to be related to a family of allergen proteins in Rosaceous plants, some members of which are associated with pathogenesis-related proteins. The amino acid sequence of the 16 kDa protein appeared to have no homology with any proteins in the SwissProt database. Therefore, it is likely that the 16 kDa protein, in a strict sense, is a bark storage protein. Defining storage proteins solely by their pattern of accumulation and the extent to which they accumulate may not be a good functional definition. It is possible that storage proteins have functional roles in addition to nitrogen storage.     

Positive association between understory species richness and a dominant shrub species (Corylus avellana) in a boreonemoral spruce forest

Old growth stands of boreonemoral spruce (Picea abies) forests frequently have a shrub layer dominated by hazel (Corylus avellana) – a species which is generally excluded in intensively managed forests due to clearcutting activities. We sampled understory species composition, richness and biomass, as well as environmental variables beneath these two species and also within forest ‘gaps’ in order to determine the effect of overstory species on understory vegetation. Species richness and biomass of herbaceous plants was significantly greater under Corylus compared with plots under Picea and in forest gaps. Indicator species analysis found that many species were significantly associated with Corylus. We found 45% of the total species found under woody plants occurred exclusively under Corylus. Light availability in spring and summer was higher in gaps than under forest cover but no difference was found between plots under Corylus and Picea. Hence, reductions in light availability cannot explain the differences in species composition. However, Ellenberg indicator values showed that more light demanding species were found under Corylus compared to Picea, but most light demanding species were found in gaps. The litter layer under Picea was three times thicker than under Corylus and this may be an important mechanism determining differences in understory composition and richness between the woody species. The presence of Corylus is an important factor enhancing local diversity and small-scale species variation within coniferous stands. Hence, management should maintain areas of Corylus shrubs to maintain understory species diversity in boreal forests.     

Identification of two thaumatin-like proteins (TLPs) in the pollination drop of hybrid yew that may play a role in pathogen defence during pollen collection

We describe the proteomic identification of two pathogenesis-related group 5 (PR-5) proteins, an acidic thaumatin-like protein (TLP) and a basic TLP isolated from the pollination drop of hybrid yew (Taxus x media Rehder). The basic TLP (TxmTLPb) was the most abundant protein in the yew pollination drop based on protein spot size after two-dimensional electrophoresis. The acidic TLP (TxmTLPa) is also a major protein component of the yew ovular secretion and appears to be encoded by a number of mRNAs transcribed from a TLP gene family that has undergone limited sequence divergence. We have sequenced five acidic TLP-encoding cDNAs (TxmTLPa-1,2,3,4 and 5) isolated from the yew ovule that vary from each other by no more than five out of 233 amino acid residues in their predicted protein sequences. All of the cDNA variants encode TLPs possessing the 16 conserved cysteine residues and five charged amino acid side chains associated with antifungal activity. Amplification of genomic DNA with TxmTLPa primers indicated that at least 11 acidic TLPs with highly similar amino acid sequences may be expressed in yew tissues. Antibodies against TLPs confirmed the identity of TxmTLPa and TxmTLPb in the yew pollination drop and detected TLPs in the ovular secretions of four other species from three other conifer families. Our results suggest that TLPs are a conserved component of conifer ovular secretions and are involved in broad spectrum pathogen defence of ovules.     

瞬时过表达ThCBL4基因提高刚毛柽柳耐盐能力

[目的]本研究拟从刚毛柽柳(Tamarix hispida)中克隆获得1个ThCBL4基因,研究该基因的耐盐功能。[方法]以Calcineurin B-like protein作为关键词,对实验室前期构建的刚毛柽柳转录组序列进行比对查找获得ThCBL4基因c DNA序列,并通过RT-PCR和测序验证克隆获得的ThCBL4基因序列。利用生物信息学工具进行序列分析。利用Real time RT-PCR分析了0.4 mol·L-1NaCl、20%(w/v)PEG6000和100μmol·L-1ABA胁迫处理后ThCBL4基因在刚毛柽柳根和叶中的表达情况。为了进一步分析ThCBL4基因的耐盐功能,构建了植物过表达载体pROKⅡ-ThCBL4,抑制表达载体p FGC5941-ThCBL4,并进行柽柳瞬时转化,同时以pROKII瞬时侵染柽柳作为对照。分析比较NaCl胁迫后瞬时转化柽柳POD、SOD酶活性、MDA含量和DAB、NBT及Evans blue染色情况,以综合评定ThCBL4基因的耐盐功能。[结果]ThCBL4基因c DNA长度为1 432 bp,编码区长度675 bp,编码224个氨基酸,编码蛋白分子量为25.57 k Da,理论等电点为5.0。3种不同环境处理后,ThCBL4基因在刚毛柽柳根中均为上调表达,在刚毛柽柳叶中均为下调表达。对盐胁迫后瞬时表达柽柳ThCBL4基因的表达分析显示,成功获得瞬时过表达(标记为OE)、抑制表达(标记为RNAi)转基因株系。进一步的生理指标和生理染色结果显示,盐胁迫下过表达ThCBL4植株的保护酶类SOD与POD活性明显高于对照和抑制表达株系。NBT和DAB染色结果显示,过表达植株的O·-2和H_2O_2含量明显低于对照,抑制表达植株的O·-2和H_2O_2含量明显高于对照;而Evans blue染色和MDA含量测定结果显示,与对照相比,过表达ThCBL4植株着色更浅、MDA含量更低,而抑制表达ThCBL4植株着色更深、MDA含量更高。[结论]ThCBL4基因可能参与了刚毛柽柳的耐盐胁迫应答,瞬时过表达ThCBL4基因通过提高植株的保护酶活性,降低活性氧氧化程度和细胞损伤的程度,从而提高了转基因刚毛柽柳的耐盐能力。     

Effects of near-lethal heat stress on bud break, heat-shock proteins and ubiquitin in dormant poplar (Populus nigra Charkowiensis x P. nigra incrassata)

We assessed the effects of near-lethal heat stress on bud break, heat-shock proteins (HSPs) and ubiquitin in hybrid poplar (Populus nigra (L.) Charkowiensis x P. nigra (L.) incrassata). Shoots, with 10-15 buds each, were collected from September to March and exposed to temperatures between 20 and 60 degrees C for 2 h. Shoots were then placed in a greenhouse at 18-22 degrees C with supplemental light and cumulative bud break was recorded over a 4-week period. Samples of bud tissues were collected during and up to 96 h after heat treatment for protein analysis. De novo synthesis of proteins was monitored by exposing excised buds to [(35)S]-methionine for 3 h before, during, or after heat treatment. Heat treatments of 40-45 degrees C resulted in both a release from endodormancy and a decrease in thermal units needed for bud break during ecodormancy. The response to near-lethal heat stress was complex and was affected by intrinsic thermal sensitivity. Heat treatments were least effective during August and became progressively more effective as endodormancy progressed. In the later stages of ecodormancy, a heat treatment of 45 degrees C either inhibited bud break or killed the buds. Although temperatures of 42.5 to 45 degrees C inhibited incorporation of [(35)S]-methionine into proteins for at least 48 h, several HSPs were synthesized in response to temperatures of 40-45 degrees C. Immunoblots indicated that one of the heat-induced proteins was immunologically related to HSP70. Increases in free and conjugated forms of ubiquitin were also observed in response to heat treatment. Production of HSPs and ubiquitin, however, was not consistently associated with the heat treatments that induced the highest percentage of bud break. The roles of heat-induced protein degradation, HSPs, and ubiquitin in overcoming dormancy by near-lethal heat stress are discussed.     

一种制备(3R,4R)-4,7,7-三甲基-6-氧杂二环[3.2.1]辛烷-3,4-二醇的新方法

在催化剂过氧磷钨酸十二烷基吡啶盐(Cat-PW4)的作用下,α-蒎烯与H2O2反应生成主要产物(3R,4R)-4,7,7-三甲基-6-氧杂二环[3.2.1]辛烷-3,4-二醇。不同反应条件对反应转化率和选择性的实验结果表明,最佳反应条件为:12.8 mmolα-蒎烯、5 m L溶剂三氯甲烷、0.2 g催化剂、3.3 m L 30%H2O2,反应温度40℃,反应时间3 h,α-蒎烯转化率和产物的选择性分别为94.7%和39.8%。反应结束后,该产物存在于水相和有机相中,通过萃取和重结晶分离提纯,得率11%,纯度达到98%;其分子结构通过红外光谱、紫外光谱、1H核磁共振谱、13C核磁共振谱、低分辨率质谱及高分辨率质谱确证。     

Competition for light, water and nitrogen in an association of hazel (Corylus avellana L.) and cocksfoot (Dactylis glomerata L.)

Competition for light, water and nitrogen between hazel trees and cocksfoot grown under the trees was studied in two hazel plantations managed in different ways. The first plantation with a light transmission of at least 70% at grass level was compared with two control hazel and cocksfoot monocrops. The soil was a calcareous heavy clay, 1.4 m deep. Water stress during summer was severe in the intercropped stand as well as the grass- only plots, while it was much less in the sole stand of trees (without grass). Hazel tree water potential in the intercropped stand improved in the fourth year as the trees extended their roots similarly to the grass-free ones, despite their much lower canopy growth rate. The fourth year, local N fertilisation began to be effective as evidenced by the N content per leaf area unit and growth of the intercropped trees: grass root competition was high and root barriers were needed to exclude it. The second plantation was managed on a deep soil with a water table at approximately 2.5 m depth. The trees did not suffer from the presence of grass, and allowed a 15 to 75% light transmission rate. A control monocropped grass plot was established for comparison. On the basis of the multiple limitation hypothesis, a grass dry matter production model was built which accounts for radiation transmitted to the understorey, water supply, temperature and air moisture deficit in interaction with the radiation transmission rate (r2 = 0.716; 590 observations). The limits of such models are discussed with regard to insufficient knowledge on root dynamics. This revised version was published online in June 2006 with corrections to the Cover Date.     

HPLC法检测桔皮中橙皮甙的稳定性研究

以高效液相色谱法检测方法,对橙皮甙的稳定性进行了初步研究。结果表明：橙皮甙的耐光、耐热性能好,在碱性较强的条件下稳定存在,pH值为12时,其在溶液中的溶解度最大。     

Modelling agroforestry systems of cacao (Theobroma cacao) with laurel (Cordia alliodora) or poro (Erythrina poeppigiana) in Costa Rica

Predictive models were developed for Cordia alliodora branch and Theobroma cacao branch or leaf biomass,based on branch basal areas (r² 0.79) but the model of C. alliodora leaf biomass, although significant, was of very low accuracy (r² = 0.09) due to annual leaf fall. At age 10 years, shade tree stem biomass accounted for 80% of the total above-ground biomass of either tree. However, between the ages of 6 and 10 years, the biomass increment of T. cacao branches (3–4t.ha^–1.a^–1) was similar to that of the shade tree stems. During the same period, the net primary productivity was 35 and 28 t.ha^–1.a^–1, for the Erythrina poepigiana and and C. alliodora systems, respectively.Cocoa production under either of the shade trees C. alliodora or E. poeppigiana was 1000 kg.ha^–1.a^–1 (oven-dry; ages 6–10 yr). During the same period, C. alliodora timber production was 9 m³.ha^–1.a^–1 whilst the leguminous shade tree E. poeppigiana does not produce timber. Litterfall over the same 5 years, including crop and/or shade tree pruning residues, averages 11 and 23 t.ha^–1.a^–1, respectively. The main difference was due to E. poeppigiana pruning residues (10t.ha^–1.a^–1).Soil organic material reserves (0–45 cm) increased over 10 years from 198 to 240 t.ha^–1 in the E. poeppigiana plots and from 168–184 t.ha^–1 in the C. alliodora plots. These values, together with the productivity indices presented, provide evidence that the systems are sustainable.For economic reasons, the use of C. alliodora is recommended under the experimental conditions. however, on less fertile soils without fertilization, the greater biomass and hence nutrient return to the soil surface under E. poeppigiana, might make this the preferable shade tree.     

Variation in the reproductive performance ofEpiricania melanoleuca (Fletcher) (Lep., Epipyropidae) in relation to stage and sex of the hostPyrilla perpusilla (Walker) (Hem., Lophopidae) during rearing

Rearing ofEpiricania melanoleuca (Fletcher) on adult females ofPyrilla perpusilla (Walker) augmented the productivity of the former, which is an effective nymphal and adult ectoparasitoid of the latter, in terms of yield of total and viable eggs. The applied value of this finding in field biocontrol program is highlighted.

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Zusammenfassung Wurden die ektoparasitischen Raupen vonEpiricania melanoleuca auf adulten Weibchen der Zuckerrohr-Zikade,Pyrilla perpusilla aufgezogen, vergrößerte sich die Zahl und Lebensfähigkeit der Eier, die von den daraus resultierenden Faltern abgelegt wurden. Das Ergebnis wird im Hinblick auf einen Einsatz des Parasitoiden zur biologischen Bekämpfung des Schädlings erörtert.

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