The use of the S haplotype-specific F-box protein gene,SFB, as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot (Prunus mume)

Japanese apricot (Prunus mume) exhibits the S-RNase-based gametophytic self-incompatibility system as do other self-incompatible Prunus species. This report identifies the S haplotype-specific F-box protein gene (SFB), a candidate gene for pollen-S, of Japanese apricot, which leads to the development of a molecular typing system for S-haplotype in this fruit species. Both 5- and 3-RACE (rapid amplification of cDNA ends) were performed with SFB gene-specific oligonucleotide primers to clone Pm-SFB¹ and Pm-SFB⁷ of 'Nanko (S¹S⁷)'. As in the case of SFB of other Prunus species, Pm-SFB¹ and Pm-SFB⁷ showed a high level of S-haplotype-specific sequence polymorphism and their expression was specific to pollen. Genomic DNA-blot analyses of 11 Japanese apricot cultivars with the Pm-SFB probes under low stringency conditions yielded RFLP bands specific to the S¹- to S⁸-haplotypes as well as a self-compatible S^f-haplotype. A practical usage of SFB as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot is discussed.Communicated by H.F. LinskensThe nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank/DDBJ database under accession numbers, AB101440 and AB101441, for SFB¹ and SFB⁷, respectively     

Genotype-dependent differences in S12-RNase expression lead to sporadic self-compatibility

Sporadic self-compatibility, the occasional fruit formation after otherwise incompatible pollinations, has been observed in some S ₁₂-containing genotypes of Solanum chacoense but not in others. We have sequenced this S ₁₂ allele and analyzed its expression in four different genotypes. The S₁₂-RNase levels were generally less abundant than those of other S-RNases present in the same plants. In addition, two-fold and five-fold differences in the amount of S₁₂-RNase and S ₁₂ RNA, respectively, were observed among the genotypes analyzed. A comparison with the genetic data showed that genotypes with the highest levels were fully and permanently self-incompatible, whereas those with the lowest levels were those in which sporadic self-compatibility had been observed. The mature protein contains four potential glycosylation sites and genotype-specific differences in the pattern of glycosylation are also observed. Our results suggest the presence of modifier genes which affect, in a genotype-dependent manner, the level of expression and the post-translational modification of the S₁₂-RNase.     

Comparative proteomic analysis of pistil abortion in Japanese apricot (Prunus mume Sieb. et Zucc)

The phenomenon of pistil abortion widely occurs in Japanese apricot and has seriously affected the yield in production. We used a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) approaches to identify the differentially expressed proteome between perfect and imperfect flower buds in Japanese apricot. More than 400 highly reproducible protein spots (P<0.05) were detected and 27 protein spots showed a greater than two-fold difference in their expression values. The proteins identified were classified into eight functional classifications and ten process categories, according to the Gene Ontology (GO). Acetyl-CoA produced by ATP citrate lyase (ACL) as a structural substance during formation of the cell wall could regulate pistil abortion in Japanese apricot. S-adenosylmethionine (SAM), xyloglucan endotransglucosylase/hydrolases (XTHs) and caffeoyl-CoA-O-methyl transferase (CCoAOMT) could promote cell wall formation in perfect flower buds of Japanese apricot, greatly contributing to pistil development. Spermidine hydroxycinnamoyl transferase (SHT) may be involved in the O-methylation of spermidine conjugates and could contribute to abnormal floral development. The identification of such differentially expressed proteins provides new targets for future studies that will assess their physiological roles and significance in pistil abortion.     

Molecular characterization of S-RNase genes and S-genotypes in the Japanese apricot (Prunus mume Sieb. et Zucc.)

Three partial S-RNase genes, MSRN-1, MSRN-2, and MSRN-3, in the Japanese apricot (Prunus mume Sieb. et Zucc.) were isolated from the three cultivars Nankou, Gyokuei, and Kairyouuchidaume, respectively. The structural characteristics revealed that S-RNase genes from the Japanese apricot were in the T2/SRNase-type S-RNase family with five conserved regions (C1, C2, C3, RC4, and C5) and one variable region (RHV) as reported in the other rosaceous plants. In the phylogenetic tree of T2/S SRNase-type RNases, three S-RNase genes of the Japanese apricot did not form a species-specific subgroup but the Prunus subfamily did. At least seven S-allelic genes were present in the Japanese apricot, and S-genotypes of six representative cultivars, including Nankou, Gyokuei, Kairyouuchidaume, Baigou, Kagajizou, and Oushuku were first established in this study as S ₁ S ₇, S ₂ S ₆, S ₃ S ₄, S ₃ S ₆, S ₃ S ₆ and S ₁ S ₅, respectively. An extended elucidation of the S-genotype would contribute to a more efficient breeding program of the Japanese apricot. Received: 5 September 2000 / Revision accepted: 22 December 2000     

Negative effects of hydroxyl radical-generating mists (simulated dew water) on the photosynthesis and growth of Japanese apricot seedlings (Prunus mume)

The hydroxyl (OH) radical, which is generated in polluted dew water on leaf surfaces of the Japanese apricot (Prunus mume), is known to be a potent oxidant. In order to investigate the effects of the OH radical formed in polluted dew water on the photosynthesis and growth of 3-year-old seedlings of P. mume, OH radical-generating solutions simulating polluted dew water were sprayed in the early morning as a mist throughout a growing season onto the leaf surfaces of seedlings growing in experimental greenhouses. Four OH radical-generating solutions (0, 6, 18 and 54 M H₂O₂ with Fe(III) and an oxalate ion) were used in the mist treatment. Five months after the beginning of treatment, the leaves exposed to the mist containing 54 M H₂O₂ showed a significantly smaller maximum CO₂ assimilation rate (A_max) and stomatal conductance (g_s) as compared to the leaves exposed to the mist containing 0 M H₂O₂. Exposure of P. mume seedlings to the OH radical-generating mist had caused a reduction in the dry weight and relative growth rate (RGR) of the above-ground parts (stem + branch) at the end of the growing season. A significant positive correlation was shown between RGR and A_max. Thus, the effects of oxidants generated in polluted dew water on leaf surfaces can be considered to be a cause of the decrease in leaf photosynthesis and growth of P. mume.     

Origin and dissemination of the pollen-part mutated SC haplotype which confers self-compatibility in apricot (Prunus armeniaca)

In China, its centre of origin, apricot (Prunus armeniaca) is self-incompatible. However, most European cultivars are self-compatible. In most cases, self-compatibility is a result of a loss-of-function mutation within the pollen gene (SFB) in the SC haplotype. Controlled pollinations performed in this work revealed that the cross 'Ceglédi óriás' (S8S9)x'Ceglédi arany' (SCS9) set well, as expected, but the reciprocal cross did not. Apricot S8, S9 and SC haplotypes were analysed using a multilevel approach including fruit set evaluation, pollen tube growth analysis, RNase activity assays, polymerase chain reaction (PCR) analysis and DNA sequencing of the S-RNase and SFB alleles. SFB8 was revealed to be the first known progenitor allele of a naturally occurring self-compatibility allele in Prunus, and consequently SC=The first intron of SC-RNase is a phase one intron, indicating its more recent evolutionary origin compared with the second intron. Sequence analysis of different cultivars revealed that more single nucleotide polymorphisms accumulated in SC-RNase than in SFBC. New methods were designed to allow high-throughput analysis of S genotypes of apricot cultivars and selections. S-RNase sequence data from various sources helped to elucidate the putative origin and dissemination of self-compatibility in apricot conferred by the SC haplotype.     

Inheritance of resistance to Plum pox virus in apricot (Prunus armeniaca L.)

The inheritance of resistance to Plum pox virus (PPV) has been studied in 1,178 apricot hybrids. Seven hundred and eighteen F1 hybrids, obtained from controlled crosses between the susceptible Greek cultivar “Bebecou” and the resistant PPV cultivars of American origin (“Stark Early Orange,” ‘NJA2,” ‘Veecot,” “Sunglo,” “Harlayne,” and “Orangered”) were evaluated for resistance to the PPV-M (Marcus) strain, 8 years after artificial inoculation. The inheritance of resistance to PPV has been additionally studied for the first time in a BC₁ population of 95 apricot hybrids for four vegetative periods. Reaction of each hybrid to PPV-M was scored through visual symptoms, indexing onto GF-305 and double-antibody sandwich enzyme-linked immunosorbent assay tests. Segregation within the hybrids, determined by Chi-squared analysis, fits a 1:1 ratio (P ≤ 0.05) of the resistant vs susceptible, indicating that resistance to PPV is controlled by a single dominant gene locus and that the above six resistant cultivars are heterozygous for the trait. Plants carrying this gene may initially develop disease symptoms on leaves but eventually recover and no virus can be detected in leaves. Susceptible plants show similar symptoms initially but remain symptomatic. Inheritance of resistance to PPV also has been studied in 365 F1 hybrids by crossing the resistant cultivar “Stella” with the susceptible “Bebecou” and the resistant cultivars “Sunglo” and “NJA2,” for 8 years after inoculation. The segregation ratio was 1:0 (resistant/susceptible) suggesting that “Stella” is homozygous for the resistance trait. The purpose of this work was the enhancement of the knowledge of inheritance of resistance to PPV for the selection of new cultivars.     

Sequence Analysis of New S-RNase and SFB alleles in Japanese Apricot (Prunus mume)

As with other self-incompatible Prunus species, cultivars of Japanese apricot (Prunus mume Sieb. et Zucc.) display the S-RNase-based gametophytic self-incompatibility system. In this study, S-genotypes of ten Japanese apricot cultivars native to China were subjected to polyacrylamide gel electrophoresis (PAGE) analysis using an efficient Prunus S-RNase primer pair, Pru-C2 and PCE-R. In addition, three new S-RNase genes (S ₃₄ , S ₃₅ and S ₃₆ -RNase) and six new SFB genes (PmSFB14, PmSFB18, PmSFB22, PmSFB24, PmSFB31 and PmSFB34) were identified and their sequences were characterized and deposited in the GenBank database.     

miR169 and PmRGL2 synergistically regulate the NF-Y complex to activate dormancy release in Japanese apricot (Prunus mume Sieb. et Zucc.)

Plant Molecular Biology - This study is the first to demonstrate that GA4-induced dormancy release is associated with the NF-Y complex, which interacts with gibberellin inhibitor RGL2 in Japanese...     

Characterization of a new (R)-hydroxynitrile lyase from the Japanese apricot Prunus mume and cDNA cloning and secretory expression of one of the isozymes in Pichia pastoris

PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.     

Inheritance of self-compatibility in almond: breeding strategies to assure self-compatibility in the progeny

To assure self-compatibility in the progenies, three different crosses were conducted for the first time in an almond breeding programme: self-pollination (266 descendants from 30 families), crosses between parents sharing an S-allele (108 descendants from five families) and crosses with homozygous self-compatible parents (62 descendants from five families). Depending on the cross, self-compatibility in the progenies was determined by observing pollen tube growth (by means of fluorescence microscopy), stylar S-RNases analysis or allele-specific PCR. The results obtained fit with the accepted hypothesis of inheritance of self-compatibility and the three crossing strategies used ensured 100% of self-compatible descendants. These strategies increase the efficiency of the breeding programme and avoid the laborious task of evaluating this characteristic. From the breeding point of view, self-fertilisation and crosses between relatives tend to produce inbreeding. Furthermore, these methods reduce the possibilities of choosing the parental combination. The use of homozygous self-compatible parents does not have any of these disadvantages. As far as we know, this is the first time that allele-specific PCR has been used for early selection of self-compatible seedlings. The advantages and disadvantages of the three methodologies used to determine self-compatibility are discussed.     

Lignin is linked to ethyl-carbamate formation in ume (Prunus mume) liqueur

Ethyl carbamate concentrations in oak barrel-aged ume (Prunus mume) liqueurs were measured, and possible explanations for elevated levels were examined. The average concentration was 0.30 mg/L, significantly higher than in ume liqueurs not aged in oak (0.08 mg/L). Oak powder extracts were prepared from both untoasted and toasted oak powder by extraction with aqueous ethanol, and these were used to make ume liqueurs. Relative to a no-oak control, the ethyl carbamate concentrations were 3.8 and 11 times higher in the ume liqueur made with the untoasted and toasted oak powder extracts respectively. The extracts were loaded onto a C18 column, washed with water, and eluted with methanol. The (13)C-NMR spectra for the main constituents of the methanol elution fractions were consistent with those for lignin or fragments thereof. The methanol fractions were added to ume liqueur which was stored for 3 months. Relative to a control, the ethyl carbamate concentrations in the 3-month old liqueurs were found to be 1.2 and 4.6 higher for the untoasted oak-powder and the toasted oak-powder respectively. Ethyl carbamate was formed when lignin was added to a 40% aqueous ethanol solution that contained potassium cyanide. These observations suggest that lignin or fragments thereof promote the formation of ethyl carbamate.     

Stamen development and winter dormancy in apricot (Prunus armeniaca)

Background and Aims

In temperate woody perennials, flower bud development is halted during the winter, when the buds enter dormancy. This dormant period is a prerequisite for adequate flowering, is genetically regulated, and plays a clear role in possibly adapting species and cultivars to climatic areas. However, information on the biological events underpinning dormancy is lacking. Stamen development, with clear differentiated stages, appears as a good framework to put dormancy in a developmental context. Here, stamen developmental changes are characterized in apricot (Prunus armeniaca) and are related to dormancy.

Methods

Stamen development was characterized cytochemically from the end of August to March, over 4 years. Developmental changes were related to dormancy, using the existing empirical information on chilling requirements.

Key Results

Stamen development continued during the autumn, and the flower buds entered dormancy with a fully developed sporogenous tissue. Although no anatomical changes were observed during dormancy, breaking of dormancy occurred following a clear sequence of events. Starch accumulated in particular places, pre-empting further development in those areas. Vascular bundles developed and pollen mother cells underwent meiosis followed by microspore development.

Conclusions

Dormancy appears to mark a boundary between the development of the sporogenous tissue and the occurrence of meiosis for further microspore development. Breaking of dormancy occurs following a clear sequence of events, providing a developmental context in which to study winter dormancy and to evaluate differences in chilling requirements among genotypes.     

Ovary starch reserves and flower development in apricot (Prunus armeniaca)

In histerant species where flowering takes place prior to leaf emergence, a flower lifespan occurs in the absence of new photoassimilates and at the expense of pre-stored reserves either in the plant as a whole or in the flower itself. In the present study, the role that the photoassimilates stored in the flowers might play in flower development from anthesis to fertilization in Prunus armeniaca L. (apricot), a histerant species, was explored. Starch content in individual flowers was measured with the help of an image analysis system. Starch content decreased from its highest value at anthesis and disappeared from the ovary 9 days later. This decrease was inversely related to an increase in ovary size and in cell number in the pericarp, suggesting an intraflower, self-supported development. This process is conserved in both pollinated and nonpollinated flowers and therefore seems to be inherent to the flower at anthesis. The onset of fruiting is preceded by the establishment of large differences among ovaries; while some experience continuous growth, others stop growing and eventually drop. Interestingly, large differences in starch content are found among flowers at anthesis. These results are discussed in terms of the possible implications of pre-stored starch in the flower supporting initial flower development.     

Identification and Characterisation of SFBs in Prunus mume

The gametophytic self-incompatibility system in Rosaceae is controlled by a single highly polymorphic gene complex, termed the S locus, which is comprised of tightly linked stylar-expressed gene, S-RNase, with a pollen-expressed gene. In this study, seven novel S haplotype-specific F-box protein genes, PmSFB ₁₀ to PmSFB ₁₆ in Prunus mume, have been identified and characterised. Similarities amongst the deduced amino acid sequences of these SFBs ranged from 73.2% to 90.9%. Comparisons of two trans-specific pairs of haplotypes, PmS ₁₁ with PspS _3-1 and PmS ₁₃ /ParS ₉ with PspS ₈ , indicated high degrees of polypeptide sequence identity. The deduced amino acid sequences of PmSFB ₁₁ and PspSFB _3-1 showed similarity scores of 99.1%. In addition, the deduced amino acid sequences of the corresponding S-RNases, PmS ₁₁ -RNase and PspS _3-1 -RNase exhibited a high similarity score of 99.0%. The similarity scores of the sequences physically positioned between S-RNase and SFB in the PmS ₁₁ haplotype and PspS _3-1 were 94.6%. The deduced amino acid sequence of PmSFB ₁₃ showed high similarity scores with those of PspSFB ₈ and ParSFB ₉ at 99.3% and 97.9%, respectively. At the deduced amino acid level, the similarity scores of the PmS ₁₃ -RNase with the PspS ₈ -RNase or the ParS ₉ -RNase were 99.0% or 99.1%, respectively. Moreover, the similarity score of the sequence separating S-RNase and SFB in PmS ₁₃ was 96.1% compared with that of the PspS ₈ haplotype and 99.0% compared with that of the ParS ₉ haplotype. Our results suggest that the S haplotypes in P. mume, P. armeniaca and P. spinosa may have a common ancestor.     

Flower bud differentiation and development in fruiting and non-fruiting shoots in relation to fruit set in apricot (Prunus armeniaca L.)

Situations of high flower bud drop and low fruit set without apparent causes are common in fruit trees. The term flower quality has been coined to explain differences among flowers in their capacity to set fruit, but the causes underpinning these differences are largely unknown. This lack of knowledge is based on the fact that these differences are established a posteriori and there are no criteria to determine a priori what will make a flower to set a fruit or to drop. In this work, we profit from the empirical knowledge that there are fruiting and non-fruiting shoots to explore to which extent flower bud differentiation and bud development will affect the subsequent fruit set. For this purpose, the processes from flower bud differentiation to fruit set were sequentially analyzed in both types of shoots, over 2 years. More than half of the buds from long shoots aborted development and dropped before flowering. At anthesis, most of the remaining flowers showed underdeveloped pistils that failed to sustain pollen germination or pollen tube growth along the pistil. This unsuccessful development resulted in clear differences in fruit set between both types of branches. These results highlight that flower bud differentiation and development play an important role for fruit set and that developmental timing appears critical to reach anthesis with a fully developed pistil.     

Genome-wide identification and analysis of late embryogenesis abundant (LEA) genes in Prunus mume

Late embryogenesis abundant (LEA) proteins play important roles in plant desiccation tolerance. In this study, 30 LEA genes were identified from Chinese plum (Prunus mume) through genome-wide analysis. The PmLEA genes are distributed on all Chinese plum chromosomes except chromosome 3. Twelve (40 %) and five PmLEA genes are arranged in tandem and segmental duplications, respectively. The PmLEA genes could be divided into eight groups (LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, PvLEA18, dehydrin and seed maturation protein). Ten gene conversion events were observed and most of them (70 %) were identified in dehydrin group. Most PmLEA genes were highly expressed in flower (22/30) and up-regulated by ABA treatment (19/30).     

Characterization and mapping of non-S gametophytic self-compatibility in sweet cherry (Prunus avium L.)

Self-incompatibility in Prunus (Rosaceae) species, such as sweet cherry, is controlled by a multiallelic locus (S), in which two tightly linked genes, S-RNase and SFB (S haplotype-specific F-box), determine the specificity of the pollen and the style. Fertilization in these species occurs only if the S-specificities expressed in the pollen and the pistils are different. However, modifier genes have been proposed to be necessary for a full manifestation of the self-incompatibility response. 'Cristobalina' is a spontaneous self-compatible sweet cherry cultivar that originated in Eastern Spain. Previous studies with this genotype suggested that pollen modifier gene(s), not linked to the S-locus, may be the cause of self-incompatibility breakdown. In this work, an F(1) population from 'Cristobalina' that segregates for this trait was used to identify molecular markers linked to self-compatibility by bulked segregant analysis. One simple sequence repeat (SSR) locus (EMPaS02) was found to be linked to self-compatibility in this population at 3.2?cM. Two additional populations derived from 'Cristobalina' were used to confirm the linkage of this marker to self-compatibility. Since EMPaS02 has been mapped to the sweet cherry linkage group 3, other markers located on the same linkage group were analysed in these populations to confirm the location of the self-compatibility locus.     

鼠类对山杏（Prunus armeniaca）种子扩散及存活作用研究

虽然有关鼠类搬运森林种子的证据已很清楚,但这些初移走种子的存活情况却知之甚少。提出了一个新的标记和跟踪种子的方法－－标签法,即将种子拴一带有编码的细长金属片,研究了北京东灵山地区山杏（Prunus armeniaca）种子的扩散距离和存活率。于1998年6月19－20日,7月3日和10月23日共在24个样点释放1440粒山杏种子。几科所有释放的种子在10d内被鼠类取走,夏天释放的种子比秋天释放的种子消失的速度快。大多数种子的扩散距离在20m以内,小于鼠类的活动距离,鼠类吃掉种子的速度很快,但当种子变得稀少时,种子存活率有所提高。山杏种子6、7月份的每日存活率小于其它月份的每日存活率。