Ripening-related changes in the cell walls of Spanish pear (Pyrus communis)

Unripe Spanish pears ( Pyras commanis L. ev. Blanquilla ) were ripened at 18°C for 5 and 10 days. Softening of the cortical tissues was associated with swelling of parenchyma cell walls from 1 to more than 5 μm in 10 day ripe pears, by which time the pears were over ripe. However, there was little indication of cell separation and the middle lamella could be detected between most cell walls. Furthermore, cell separation was constrained by regions rich in plasmodesmata where wall swelling was prevented. Parenchyma cells in the 500 μm of tissue underlying the epidermis did not undergo ripening-related changes to the same extent as those of the cortex. These cells, in combination with a sub-epidermal layer of lignified sclereid clusters, constituted a relatively tough and protective skin. Ripening of the cortical tissues was associated with a depletion of alcohol-insoluble pectic polysaccharides, as indicated by the decrease in arabinose and uronic acid. Analysis of alcohol-insoluble cell wall preparations enriched in either parenchyma or sclereid cell walls indicated that this change was predominantly associated with the parenchyma walls. Such changes were less prominent in the peel. The decrease in pectic polysaccharides was accompanied by an increase in their solubility. During ripening, the sclereid clusters of the cortex continued in develop, as indicated by an increase in their size and yield of cell wall xylose and glucose. Cortical parenchyma cells radiating from the sclereids were firmly attached to the lignified cells. This was due to lignification extending from the sclereids into the primary walls of the parenchyma cells. We conclude that dissolution of pectic polysaccharides is one of several factors which determine softening during ripening of Spanish pears.     

Isolation and characterization of polymorphic microsatellite markers from European pear (Pyrus communis L.)

This study reports the development and characterization of 19 microsatellite primer pairs developed from genomic DNA of European pear (Pyrus communis) and their transferability to other Pyrus and Malus material. The primers were designed from two different genomic libraries enriched for di‐ and trinucleotide repeats. When tested in six P. communis cultivars and 15 other Pyrus species, 13 primers revealed single‐locus polymorphism and six showed more complex patterns that suggest multiple loci. Two to 18 alleles were detected per locus and two primer pairs were sufficient to discriminate all accessions. Transferability of nine primer pairs to Malus was demonstrated through amplification of discrete products in two accessions.     

Photosystem II efficiency and mechanisms of energy dissipation in iron-deficient,field-grown pear trees (Pyrus communis L.)

The dark-adapted Photosystem II efficiency of field-grown pear leaves, estimated by the variable to maximum chlorophyll fluorescence ratio, was little affected by moderate and severe iron deficiency. Only extremely iron-deficient leaves showed a decreased Photosystem II efficiency after dark adaptation. Midday depressions in Photosystem II efficiency were still found after short-term dark-adaptation in iron-deficient leaves, indicating that Photosystem II down-regulation occurred when the leaves were illuminated by excessive irradiance. The actual Photosystem II efficiency at steady-state photosynthesis was decreased by iron deficiency both early in the morning and at midday, due to closure of Photosystem II reaction centers and decreases of the intrinsic Photosystem II efficiency. Iron deficiency decreased the amount of light in excess of that which can be used in photosynthesis not only by decreasing absorptance, but also by increasing the relative amount of light dissipated thermally by the Photosystem II antenna. When compared to the controls, iron-deficient pear leaves dissipated thermally up to 20% more of the light absorbed by the Photosystem II, both early in the morning and at midday. At low light iron-deficient leaves with high violaxanthin cycle pigments to chlorophyll ratios had increases in pigment de-epoxidation, non-photochemical quenching and thermal dissipation. Our data suggest that pH could be the major factor controlling thermal energy dissipation, and that large (more than 10-fold) changes in the zeaxanthin plus antheraxanthin to chlorophyll molar ratio caused by iron deficiency were associated only to moderate increases in the extent of photoprotection.This revised version was published online in October 2005 with corrections to the Cover Date.     

Foliar fertilization to control iron chlorosis in pear (Pyrus communis L.) trees

The effectiveness of foliar fertilization to re-green chlorotic leaves in iron-deficient pear trees has been studied. Trials were made to assess the influence of (i) the level of Fe deficiency, (ii) the leaf surface treated (adaxial or abaxial), and (iii) two different surfactants, L-77 and Mistol. Treatments were ferrous sulphate alone, ascorbic, citric and sulphuric acids, applied either alone or in combination with ferrous sulphate, Fe-DTPA and water as a control. Solutions were applied with a brush and leaves were treated twice each year. None of the treatments caused a full recovery from Fe deficiency chlorosis. Treatments containing Fe caused the largest re-greening effects, and FeSO₄ had a similar re-greening effect to Fe(III)-DTPA. Increases in leaf Chl were more pronounced with abaxial leaf surface applications and in severely deficient leaves. Using Fe(III)-DTPA in foliar sprays does not seem to be justified, since their effects are not better than those of FeSO₄. The joint use of Fe(III)-DTPA and L-77 and that of FeSO₄ and citric acid do not seem to be suitable. With a single foliar application, FeSO₄ combined with acids gave slightly better results than FeSO₄ alone. Acidic solution applications without Fe may be effective in alleviating chlorosis in some cases, especially in the case of citric acid. In the current state of knowledge, foliar fertilization cannot offer yet a good alternative for full control of Fe chlorosis, although its low environmental impact and cost make this technique a good complementary measure to soil Fe-chelate applications and other chlorosis alleviation management techniques. Abbreviations: Chl – chlorophyll; EDDCHA – ethylenediamine di(5-carboxy-2-hydroxyphenylacetic) acid; EDDHA – ethylenediamine di(o-hydroxyphenylacetic) acid; EDDHMA – ethylenediamine di(o-hydroxy-p-methylphenylacetic) acid; EDDHSA – ethylenediamine di(2-hydroxy-5-sulfophenylacetic) acid     

Iron deficiency-associated changes in the composition of the leaf apoplastic fluid from field-grown pear (Pyrus communis L.) trees.

Experiments have been carried out with field-grown pear trees to investigate the effect of iron chlorosis on the composition of the leaf apoplast. Iron deficiency was associated with an increase in the leaf apoplastic pH from the control values of 5.5-5.9 to 6.5-6.6, as judged from direct pH measurements in apoplastic fluid obtained by centrifugation and fluorescence of leaves incubated with 5-CF. The major organic acids found in leaf apoplastic fluid of iron-deficient and iron-sufficient pear leaves were malate, citrate and ascorbate. The total concentration of organic acids was 2.9 mM in the controls and increased to 5.5 mM in Fe-deficient leaves. The total apoplastic concentration of inorganic cations (Ca, K and Mg) increased with Fe deficiency from 15 to 20 mM. The total apoplastic concentration of inorganic anions (Cl-, NO3-, SO4(2-) and HPO4(2-)) did not change with Fe deficiency. Iron concentrations decreased from 4 to 1.6 microM with Fe deficiency. The major Fe species predicted to exist in the apoplast was [FeCitOH](-1) in both Fe-sufficient and deficient leaves. Organic acids in whole leaf homogenates increased from 20 to 40 nmol x m(-2) with Fe deficiency. The accumulation of organic anions in the Fe-deficient leaves does not appear to be associated to an increased C fixation in leaves, but rather it seems to be a consequence of C transport via xylem.     

Rhizogenesis in callus from Conference pear (Pyrus communis L.) protoplasts

Large numbers (ca 6×10⁶ protoplasts/g f.wt) of viable (80%) protoplasts were isolated from embryo-callus tissues of Conference pear using an enzyme mixture which contained 2.0% (w/v) Meicelase, 2.0% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. A medium based on ammonium-free MS salts and supplemented with 2.0 mg/l NAA, 0.5 mg/l BAP and 9% (w/v) mannitol supported protoplast division and the proliferation of multicellular colonies. Colonies were taken to the callus stage on a medium which contained MS salts plus 0.1 mg/l 2,4-D and 0.1 mg/l BAP. Roots were regenerated from these protoplastderived calli on MS medium with 0.1 mg/l NAA, 5.0 mg/l BAP and 50 mg/l casein hydrolysate.Abbreviations BAP 6-benzylaminopurine - CPW13M CPW salts medium [15] with 13% (w/v) mannitol - FDA fluorescein diacetate, f. wt-fresh weight - MS Murashige and Skoog [14] - NAA -naphthaleneacetic acid - PE plating efficiency (%) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Adventitious shoot regeneration from in vitro leaves of several pear cultivars (Pyrus communis L.)

An efficient adventitious shoot regeneration system was developed for pear (Pyrus communis L.), using leaves from in vitro proliferating shoots. Under optimal conditions, bud regeneration frequencies of Comice, Passe-Crassane, Williams and Conference ranged from 60% to 97%, with the mean number of shoots per regenerating leaf ranging from 3.2 to 6.6. Despite the great variability in responses of the different cultivars, in general an initial dark exposure of at least 20 days was required. Ammonium and total nitrogen proved to play an essential role: intermediate NH₄ ⁺ concentrations were suitable for regeneration. The balance between NH₄ ⁺ and NO₃ ^- also influenced regeneration; optimal regeneration occured on media with a 1:3 NH₄ ⁺/NO₃ ^- ratio. TDZ at 1 M was less efficient than higher concentrations, whatever the NAA level. Finally, length and growth regulator composition of the two phases (induction and expression) influenced the regeneration rate of Conference.Abbreviations BA 6-benzyladenine - EDFS ethylenediamine-tetraacetic acid ferric-sodium salt - IBA 4-indole-3yl-butyric acid - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-ylurea)     

Inheritance and linkage of isozyme loci in pear (Pyrus communis L.)

 The polymorphism of 11 enzymes was analysed in 11 progenies from controlled crosses between pear varieties, using acrylamide and starch electrophoresis gels. Twenty-two loci were identified and segregation was scored for 20 of them. Three pairs of duplicated loci forming intergenic hybrid bands were detected, these correspond to equivalent duplicated genes in apple. A total of 49 active alleles and 1 null allele were identified. Joint segregation analysis revealed three linkage groups, which could all be related to existing groups on the apple map. The conservation of isozyme patterns, duplicated genes and linkage groups indicates a high degree of synteny between apple and pear. Received: 8 July 1996 / Accepted: 19 July 1996     

Isolation and characterization of microsatellite markers in Japanese pear (Pyrus pyrifolia Nakai)

Fourteen microsatellite markers were developed from an enriched genomic library of Japanese pear (Pyrus pyrifolia) by selective hybridization. They were characterized using 17 Japanese pear cultivars. The expected heterozygosity and observed heterozygosity ranged from 0.21 to 0.74 and from 0 to 0.88, respectively. Two to 11 alleles were detected per locus, with IPPN09 and IPPN15 judged to amplify multiple loci. IPPN17 was the most informative locus with the lowest probability of identity (0.19). These primers exhibited a high cross‐species transferability between species and genera.     

Effects of branch solid Fe sulphate implants on xylem sap composition in field-grown peach and pear: changes in Fe, organic anions and pH

The effects of placing solid implants containing Fe sulfate in branches of Fe-deficient pear and peach trees on the composition of the xylem sap have been studied. Iron sulfate implants are commercially used in northeastern Spain to control iron chlorosis in fruit trees. Implants increased Fe concentrations and decreased organic acid concentrations in the xylem sap, whereas xylem sap pH was only moderately changed. The citrate to Fe ratios decreased markedly after implants, therefore improving the possibility that Fe could be reduced by the leaf plasma membrane enzyme reductase, known to be inhibited by high citrate/Fe ratios. In peach, the effects of the implants could be observed many months post treatment. In pear, some effects were still observed one year after the implants had taken place. Results obtained indicate that solid Fe sulfate implants were capable of significantly changing the chemical composition of the xylem sap in fruit trees.     

秋水仙碱诱导梨离体叶片再生多倍体

以二倍体梨(Pyrus communis L.) 品种'丰产'试管苗的离体叶片为外植体,研究了秋水仙碱处理对叶片不定梢再生及多倍体诱导率的影响.结果表明:(1)随秋水仙碱处理时间增加,叶片不定梢再生率下降,而多倍体诱导率增加;以0.4%的秋水仙碱溶液处理叶片48 h效果最好,多倍体诱导率为6.1%.(2)流式细胞仪(Flow cytometry)分析鉴定表明,获得的多倍体有三倍体、四倍体和混倍体;多倍体与二倍体的形态特征差异明显,多倍体比二倍体茎粗、节间短,叶形指数小,根粗而且短.     

Remobilised nitrogen and root uptake of nitrate for spring leaf growth,flowers and developing fruits of pear (Pyrus communis L.) trees

Both uptake of fertiliser N and remobilisation of stored N were quantified for the early growth of spur and shoot leaves, flowers and fruit development of pear trees. One-year old Abbé F. trees grafted on quince C rootstocks were fertilised with a generous N supply for one year and while dormant during the winter, transferred to sand cultures. Each tree received 3 g of labelled nitrate-N at the end of winter and in early spring. Leaves, flowers and fruit were sampled on 5 separate occasions and the recovery of labelled N used to distinguish the remobilisation of N and the root uptake of nitrate. Remobilisation of stored N accounted for most of the N present in leaves and flowers during blossoming. Remobilisation of nitrogen stopped between petal fall and the beginning of fruit development. Root uptake of nitrate linearly increased over time and at the last sampling, 55 days after bud burst, fertiliser N contributed approximately half of the total N recovered in both spur and shoot leaves, the remainder coming from remobilisation. Flowers and fruits based their N metabolism more on remobilisation as compared to the leaves. This pattern of internal cycling of N is discussed in relation to fertilisation strategies for pear trees.     

Proteomic analysis of core breakdown disorder in Conference pears (Pyrus communis L.)

2-DE was applied to study core breakdown disorder in controlled atmosphere stored 'Conference' pears. This physiological disorder is characterized by internal browning of the fruit tissue and the development of cavities. Suitable protein phenol extraction/ammonium acetate-methanol precipitation and 2-DE protocols for a wide pH range were established for pear tissue. The protein expression profiles of healthy, sound (intact tissue of pears with core breakdown) and brown tissue were analyzed with the univariate non-parametric Kolmogorov-Smirnov test and multivariate statistical techniques such as principal component analysis and partial least square discriminant analysis. Both statistical approaches revealed interesting differentially expressed proteins between healthy and disordered pears. LC-ESI-MS/MS identification of differentially expressed proteins between healthy and sound tissue revealed their participation in the energy metabolism, the antioxidant system and ethylene biosynthesis. Up-regulated characteristic proteins in brown tissue were mainly involved in energy metabolism and defense mechanisms. Proteomics coupled to univariate and multivariate statistical techniques seems to be an efficient approach to get a better insight into the different mechanisms and pathways leading to the core breakdown disorder.     

梨多倍体化对离体叶片不定梢再生能力的影响

以源于二倍体梨品种Fertility(Pyrus communis L.)通过秋水仙碱离体诱变体细胞染色体加倍获得的不同同源多倍体无性系为试材,以离体叶片为外植体,观察研究了不同倍性无性系叶片的不定梢再生能力。结果表明,多倍体的不定梢再生率显著低于二倍体的再生率。不同多倍体无性系的不定梢再生能力也存在显著差异。三倍体无性系3x-3和四倍体无性系4x-4不能诱导产生不定梢。表明器官发生能力下降或植物细胞全能性的丧失与细胞染色体多倍体化有关。     

西洋梨水孔蛋白基因家族的全基因组鉴定及表达分析

水孔蛋白(AQPs,aquaporins)是高效转运水分子的膜内在蛋白,具有丰富的多样性,在调控植物的水分关系中有重要作用。本研究利用西洋梨(Pyrus communis L.‘Bartlett’)基因组数据库,通过生物信息学手段鉴定西洋梨PcAQPs基因家族成员;并利用MEGA 6.0.5软件,采用邻接法构建系统发育树;利用GSDS 2.0软件进行基因结构分析,MEME程序进行Motif分析,AgBase v2.00程序进行GO分析;采用半定量RT-PCR技术研究PcAQPs基因组织表达情况。结果表明,西洋梨基因组中共有54个PcAQPs家族成员,均含有AQP特征结构域和保守的Motif基序,根据基因结构及系统进化分析可分为PIP、TIP、NIP和SIP等4个亚家族。不同基因间结构差别较大,但聚类关系较近的基因其结构类似。GO分析发现,多数PcAQPs基因具有转运蛋白活性,参与物质转运、应激反应、发育和代谢等生物学过程,但不同亚家族成员构成的细胞组分及参与的生物学过程具有明显差异。半定量结果表明,大多数PcAQPs基因在根、茎、叶和果实中均有表达,而且不同基因家族、不同基因间的组织表达模式存在差异。该研究为今后西洋梨PcAQPs基因的克隆和功能分析奠定了基础。     

水分胁迫下臭柏（Sabina vulgaris Ant.）光合特性和色素组成的季节变化

在室内砾耕栽培条件下,通过培养液中加入PEG(Polyethylene glycol 分子量为6000)以调节溶液渗透势,设置对照、弱水分胁迫和强水分胁迫3种处理 (培养液渗透势分别为0.02,-0.1,-0.34 MPa), 从1997年开始对臭柏进行长期干旱胁迫模拟实验.2003年测定了臭柏叶片光合色素和光合特性的季节变化.结果表明:对照区气孔导度季节变化在5月和9月份形成了典型的双峰曲线.尽管对照区的气孔导度明显高于其他两个处理,但日光合量却低于弱水分胁迫区.3个处理Chl a/b的比值在11月至翌年3月的低温期内均升高,以强水分胁迫区的增幅最大,其它月份该比值在3个处理之间没有显著的差异.3个处理的叶绿素总量(Chl a+b)在生长季的5～9月份均有不同程度的上升,但其中以强水分胁迫区增幅最小.在11月至翌年3月的低温期,各处理均大幅提高叶黄素总量(V+A+Z)和热耗散色素比例(A+Z)/(V+A+Z) (V:紫黄质、A:单环氧玉米黄质、Z:玉米黄质);在5～7月份的生长高峰期,各处理则明显降低了叶黄素总量和热耗散色素比例.这种趋势在强水分胁迫区表现的更为显著.     

Partitioning of (13)C-photosynthate from spur leaves during fruit growth of three Japanese pear (Pyrus pyrifolia) cultivars differing in maturation date

BACKGROUND AND AIMS: In fruit crops, fruit size at harvest is an important aspect of quality. With Japanese pears (Pyrus pyrifolia), later maturing cultivars usually have larger fruits than earlier maturing cultivars. It is considered that the supply of photosynthate during fruit development is a critical determinant of size. To assess the interaction of assimilate supply and early/late maturity of cultivars and its effect on final fruit size, the pattern of carbon assimilate partitioning from spur leaves (source) to fruit and other organs (sinks) during fruit growth was investigated using three genotypes differing in maturation date. METHODS: Partitioning of photosynthate from spur leaves during fruit growth was investigated by exposure of spurs to (13)CO(2) and measurement of the change in (13)C abundance in dry matter with time. Leaf number and leaf area per spur, fresh fruit weight, cell number and cell size of the mesocarp were measured and used to model the development of the spur leaf and fruit. KEY RESULTS: Compared with the earlier-maturing cultivars 'Shinsui' and 'Kousui', the larger-fruited, later-maturing cultivar 'Shinsetsu' had a greater total leaf area per spur, greater source strength (source weight x source specific activity), with more (13)C assimilated per spur and allocated to fruit, smaller loss of (13)C in respiration and export over the season, and longer duration of cell division and enlargement. Histology shows that cultivar differences in final fruit size were mainly attributable to the number of cells in the mesocarp. CONCLUSIONS: Assimilate availability during the period of cell division was crucial for early fruit growth and closely correlated with final fruit size. Early fruit growth of the earlier-maturing cultivars, but not the later-maturing ones, was severely restrained by assimilate supply rather than by sink limitation.     

Development of an Agrobacterium-mediated transformation method for pear (Pyrus communis L.) with leaf-section and axillary shoot-meristem explants

We have developed a new Agrobacterium-mediated transformation method for the low-frequency-regenerating pear (Pyrus communis L.) cvs. Silver bell and La France. Leaf sections derived from in vitro shoots were initially used for the transformation procedure. Under optimum transformation conditions, which included culture and selection on 30 mg/l kanamycin (Km) combined with 500 mg/l sulbenicillin, a 3.2% transformation efficiency was obtained for cv. Silver bell, but no transformants of La France were obtained because of the very low regeneration frequency. Axillary shoot meristems were then examined as potential explants for La France. Selection in 5 mg/l Km and 375 mg/l carbenicillin resulted in transformed shoots being produced at an efficiency of 4.8%, and the apparent white Km-sensitive shoots were not formed during a 2-year subculture on micropropagation medium containing 50 mg/l Km. Therefore, transformations using axillary shoot meristems may be an alternative method for pear cultivars recalcitrant to regeneration from leaf sections.     

Changes in tonoplast protein and density with the development of pear fruit

Protoplasts isolated from pear fruit at the end of the cell‐division stage, 30 days after flowering (DAF), had already formed a large central vacuole and the vacuole occupied most of the protoplast. The changes in protein composition and density of the tonoplast (vacuolar membrane) were investigated during fruit development. After a linear sucrose density gradient centrifugation, the distribution of tonoplasts at 30 and 48 DAF was broad and began to narrow with further fruit development. This suggests that the tonoplast of young fruit is heterogeneous and becomes homogeneous with fruit development. The apparent density of the tonoplast at 30 DAF was approximately 1.12 g ml⁻¹; it decreased with fruit development and was finally 1.09 g ml⁻¹ in mature fruit. The phospholipid amount on the basis of tonoplast protein was 0.80 mg mg⁻¹ at 30 DAF. It increased with fruit development, and finally reached 7.49 mg mg⁻¹. This result indicates that the decrease in the density of the tonoplast was caused by the increase in the ratio of phospholipid to membrane protein. The protein composition of the tonoplast at each stage was quite different. The level of polypeptides of 94, 70, 61, 52, 48 and 41 kDa was low in young fruit and high in the middle or later stages of fruit development. In contrast, the level of a 76‐kDa polypeptide was high in young fruit and decreased with fruit development. Although their functions are still unclear, these tonoplast proteins may play important roles in fruit development.     

Iron deficiency-induced changes in carbon fixation and leaf elemental composition of sugar beet (Beta vulgaris) plants

In this experiment we (i) tested the hypothesis that, besides decreasing leaf C fixation, lime induced iron (Fe) deficiency increases root C fixation via PEP carboxylase and (ii) assessed the Fe-induced modifications in the elemental composition of plant tissues. Sugar beet plants were grown in nutrient solutions with Fe (45 M Fe-EDTA; +Fe control) or in a similar nutrient solution without Fe (–Fe) and in presence of CaCO₃ (1.0 gL^–1), either labelled with ¹³C (20 at. %) or unlabelled. After 7 and 17 days from treatment imposition, plants were harvested and single organs analysed for total O, C, H, macro and micronutrients. ¹³C abundance was also assessed in control, unlabelled and labelled –Fe plants. Iron deficiency caused significant growth reductions; chlorophyll and net photosynthesis decreased markedly in Fe-deficient plants when compared to the controls, whereas leaf transpiration rates and stomatal conductance were not affected by Fe deficiency. Iron deficient plants had leaf biomass with lower C (2 to 4%) and higher O (3 to 5%) concentrations than +Fe plants. The ¹³C was higher (less negative) in +Fe than in –Fe unlabelled plants. Iron deficient plants grown in the nutrient solution enriched with labelled CaCO₃ absorbed a relatively small amount of labelled C, which was mainly recovered in the fine roots and accounted for less than 2% of total C gain in the 10 d treatment period. Evidences suggest that iron deficient sugar beets grown in the presence of CaCO₃ do not markedly shift their C fixation from leaf RuBP to root PEPC.