Gene Expression Analysis of Induced Plum pox virus (Sharka) Resistance in Peach (Prunus persica) by Almond (P. dulcis) Grafting.

No natural sources of resistance to Plum pox virus (PPV, sharka disease) have been identified in peach. However, previous studies have demonstrated that grafting a "Garrigues" almond scion onto "GF305" peach rootstock seedlings heavily infected with PPV can progressively reduce disease symptoms and virus accumulation. Furthermore, grafting a "Garrigues" scion onto the "GF305" rootstock has been shown to completely prevent virus infection. This study aims to analyse the rewiring of gene expression associated with this resistance to PPV transmitted by grafting through the phloem using RNA-Seq and RT-qPCR analysis. A total of 18 candidate genes were differentially expressed after grafting "Garrigues" almond onto healthy "GF305" peach. Among the up-regulated genes, a HEN1 homolog stands out, which, together with the differential expression of RDR- and DCL2-homologs, suggests that the RNA silencing machinery is activated by PPV infection and can contribute to the resistance induced by "Garrigues" almond. Glucan endo-1,3-beta D-glucosidase could be also relevant for the "Garrigues"-induced response, since its expression is much higher in "Garrigues" than in "GF305". We also discuss the potential relevance of the following in PPV infection and "Garrigues"-induced resistance: several pathogenesis-related proteins; no apical meristem proteins; the transcription initiation factor, TFIIB; the speckle-type POZ protein; in addition to a number of proteins involved in phytohormone signalling.

Pedigree analysis of 220 almond genotypes reveals two world mainstream breeding lines based on only three different cultivars.

Loss of genetic variability is an increasing challenge in tree breeding programs due to the repeated use of a reduced number of founder genotypes. However, in almond, little is known about the genetic variability in current breeding stocks, although several cases of inbreeding depression have been reported. To gain insights into the genetic structure in modern breeding programs worldwide, marker-verified pedigree data of 220 almond cultivars and breeding selections were analyzed. Inbreeding coefficients, pairwise relatedness, and genetic contribution were calculated for these genotypes. The results reveal two mainstream breeding lines based on three cultivars: "Tuono", "Cristomorto", and "Nonpareil". Descendants from "Tuono" or "Cristomorto" number 76 (sharing 34 descendants), while "Nonpareil" has 71 descendants. The mean inbreeding coefficient of the analyzed genotypes was 0.041, with 14 genotypes presenting a high inbreeding coefficient, over 0.250. Breeding programs from France, the USA, and Spain showed inbreeding coefficients of 0.075, 0.070, and 0.037, respectively. According to their genetic contribution, modern cultivars from Israel, France, the USA, Spain, and Australia trace back to a maximum of six main founding genotypes. Among the group of 65 genotypes carrying the Sf allele for self-compatibility, the mean relatedness coefficient was 0.125, with "Tuono" as the main founding genotype (24.7% of total genetic contribution). The results broaden our understanding about the tendencies followed in almond breeding over the last 50 years and will have a large impact into breeding decision-making process worldwide. Increasing current genetic variability is required in almond breeding programs to assure genetic gain and continuing breeding progress.

Identification of early and late flowering time candidate genes in endodormant and ecodormant almond flower buds.

Flower bud dormancy in temperate fruit tree species, such as almond [Prunus dulcis (Mill.) D.A. Webb], is a survival mechanism that ensures that flowering will occur under suitable weather conditions for successful flower development, pollination and fruit set. Dormancy is divided into three sequential phases: paradormancy, endodormancy and ecodormancy. During the winter, buds need cultivar-specific chilling requirements (CRs) to overcome endodormancy and heat requirements to activate the machinery to flower in the ecodormancy phase. One of the main factors that enables the transition from endodormancy to ecodormancy is transcriptome reprogramming. In this work, we therefore monitored three almond cultivars with different CRs and flowering times by RNA sequencing during the endodormancy release of flower buds and validated the data by quantitative real-time PCR in two consecutive seasons. We were thus able to identify early and late flowering time candidate genes in endodormant and ecodormant almond flower buds associated with metabolic switches, transmembrane transport, cell wall remodeling, phytohormone signaling and pollen development. These candidate genes were indeed involved in the overcoming of the endodormancy in almond. This information may be used for the development of dormancy molecular markers, increasing the efficiency of temperate fruit tree breeding programs in a climate-change context.

Advancing Endodormancy Release in Temperate Fruit Trees Using Agrochemical Treatments.

Endodormancy in temperate fruit trees like Prunus is a protector state that allows the trees to survive in the adverse conditions of autumn and winter. During this process, plants accumulate chill hours. Flower buds require a certain number of chill hours to release from endodormancy, known as chilling requirements. This step is crucial for proper flowering and fruit set, since incomplete fulfillment of the chilling requirements produces asynchronous flowering, resulting in low quality flowers, and fruits. In recent decades, global warming has endangered this chill accumulation. Because of this fact, many agrochemicals have been used to promote endodormancy release. One of the first and most efficient agrochemicals used for this purpose was hydrogen cyanamide. The application of this agrochemical has been found to advance endodormancy release and synchronize flowering time, compressing the flowering period and increasing production in many species, including apple, grapevine, kiwi, and peach. However, some studies have pointed to the toxicity of this agrochemical. Therefore, other non-toxic agrochemicals have been used in recent years. Among them, Erger® + Activ Erger® and Syncron® + NitroActive® have been the most popular alternatives. These two treatments have been shown to efficiently advance endodormancy release in most of the species in which they have been applied. In addition, other less popular agrochemicals have also been applied, but their efficiency is still unclear. In recent years, several studies have focused on the biochemical and genetic variation produced by these treatments, and significant variations have been observed in reactive oxygen species, abscisic acid (ABA), and gibberellin (GA) levels and in the genes responsible for their biosynthesis. Given the importance of this topic, future studies should focus on the discovery and development of new environmentally friendly agrochemicals for improving the modulation of endodormancy release and look more deeply into the effects of these treatments in plants.

Ascorbic acid and prunasin, two candidate biomarkers for endodormancy release in almond flower buds identified by a nontargeted metabolomic study.

Temperate fruit trees belonging to Prunus species have the ability to suspend (induce dormancy) and resume growth periodically in response to environmental and seasonal conditions. Endodormancy release requires the long-term accumulation of chill. Upon accumulation of cultivar-specific chill requirements, plants enter the state of ecodormancy, which means the ability to grow has been restored, depending on the fulfilment of heat requirements. As many different metabolic pathways are implicated in endodormancy release, we have performed a metabolomic analysis, using the ultra-high-performance liquid chromatography-quadrupole time-of-flying (UPLC-QToF) technique. We assayed flower buds in different stages of endodormancy in four almond cultivars with different flowering times: the extra-early Desmayo Largueta, the late Antoñeta, the extra-late Penta, and the ultra-late Tardona. An orthogonal projection to latent-structure discriminant-analysis model was created to observe differences between endodormant and ecodormant flower buds. The metabolites showing the most significant variation were searched against the Metlin, HMDB, and KEGG libraries, which allowed us to identify 87 metabolites. These metabolites were subsequently assigned to specific pathways, such as abscisic acid biosynthesis, phenylpropanoid biosynthesis, and D-sorbitol metabolism, among others. The two metabolites that exhibited the most significant variations in all the cultivars studied with fold changes of up to 6.49 were ascorbic acid and prunasin. For the first time, these two metabolites have been proposed as potential biomarkers for endodormancy release in almond. Given the high synteny present between the Rosaceae species, these results could be extrapolated to other important crops like peach, plum, cherry, or apricot, among others.

DNA Methylation Analysis of Dormancy Release in Almond (Prunus dulcis) Flower Buds Using Epi-Genotyping by Sequencing.

DNA methylation and histone post-translational modifications have been described as epigenetic regulation mechanisms involved in developmental transitions in plants, including seasonal changes in fruit trees. In species like almond (Prunus dulcis (Mill.) D.A: Webb), prolonged exposure to cold temperatures is required for dormancy release and flowering. Aiming to identify genomic regions with differential methylation states in response to chill accumulation, we carried out Illumina reduced-representation genome sequencing on bisulfite-treated DNA from floral buds. To do this, we analyzed almond genotypes with different chilling requirements and flowering times both before and after dormancy release for two consecutive years. The study was performed using epi-Genotyping by Sequencing (epi-GBS). A total of 7317 fragments were sequenced and the samples compared. Out of these fragments, 677 were identified as differentially methylated between the almond genotypes. Mapping these fragments using the Prunus persica (L.) Batsch v.2 genome as reference provided information about coding regions linked to early and late flowering methylation markers. Additionally, the methylation state of ten gene-coding sequences was found to be linked to the dormancy release process.

Elucidation of the Amygdalin Pathway Reveals the Metabolic Basis of Bitter and Sweet Almonds (Prunus dulcis).

Almond (Prunus dulcis) is the principal Prunus species in which the consumed and thus commercially important part of the fruit is the kernel. As a result of continued selection, the vast majority of almonds have a nonbitter kernel. However, in the field, there are trees carrying bitter kernels, which are toxic to humans and, consequently, need to be removed. The toxicity of bitter almonds is caused by the accumulation of the cyanogenic diglucoside amygdalin, which releases toxic hydrogen cyanide upon hydrolysis. In this study, we identified and characterized the enzymes involved in the amygdalin biosynthetic pathway: PdCYP79D16 and PdCYP71AN24 as the cytochrome P450 (CYP) enzymes catalyzing phenylalanine-to-mandelonitrile conversion, PdUGT94AF3 as an additional monoglucosyl transferase (UGT) catalyzing prunasin formation, and PdUGT94AF1 and PdUGT94AF2 as the two enzymes catalyzing amygdalin formation from prunasin. This was accomplished by constructing a sequence database containing UGTs known, or predicted, to catalyze a ß(1→6)-O-glycosylation reaction and a Basic Local Alignment Search Tool search of the draft version of the almond genome versus these sequences. Functional characterization of candidate genes was achieved by transient expression in Nicotiana benthamiana Reverse transcription quantitative polymerase chain reaction demonstrated that the expression of PdCYP79D16 and PdCYP71AN24 was not detectable or only reached minute levels in the sweet almond genotype during fruit development, while it was high and consistent in the bitter genotype. Therefore, the basis for the sweet kernel phenotype is a lack of expression of the genes encoding the two CYPs catalyzing the first steps in amygdalin biosynthesis.

Synteny-Based Development of CAPS Markers Linked to the Sweet kernel LOCUS, Controlling Amygdalin Accumulation in Almond (Prunus dulcis (Mill.) D.A.Webb).

The bitterness and toxicity of wild-type seeds of Prunoideae is due to the cyanogenic glucoside amygdalin. In cultivated almond (Prunus dulcis (Mill.) D.A. Webb), a dominant mutation at the Sk locus prevents amygdalin accumulation and thus results in edible sweet kernels. Here, we exploited sequence similarity and synteny between the genomes of almond and peach (Prunus persica (L.) Batsch) to identify cleaved amplified polymorphic sequence (CAPS) molecular markers linked to the Sk locus. A segregant F1 population was used to map these markers on the Sk genomic region, together with Sk-linked simple sequence repeat (SSR) markers previously described. Molecular fingerprinting of a cultivar collection indicated the possibility to use CAPS polymorphisms identified in this study in breeding programs arising from different parental combinations. Overall, we highlight a set of codominant markers useful for early selection of sweet kernel genotypes, an aspect of primary importance in almond breeding. In addition, by showing collinearity between the physical map of peach and the genetic map of almond with respect to the Sk genomic region, we provide valuable information for further marker development and Sk positional cloning.

ß-Glucosidase activity in almond seeds.

Almond bitterness is the most important trait for breeding programs since bitter-kernelled seedlings are usually discarded. Amygdalin and its precursor prunasin are hydrolyzed by specific enzymes called ß-glucosidases. In order to better understand the genetic control of almond bitterness, some studies have shown differences in the location of prunasin hydrolases (PH, the ß-glucosidase that degrades prunasin) in sweet and bitter genotypes. The aim of this work was to isolate and characterize different PHs in sweet- and bitter-kernelled almonds to determine whether differences in their genomic or protein sequences are responsible for the sweet or bitter taste of their seeds. RNA was extracted from the tegument, nucellus and cotyledon of one sweet (Lauranne) and two bitter (D05-187 and S3067) almond genotypes throughout fruit ripening. Sequences of nine positive Phs were then obtained from all of the genotypes by RT-PCR and cloning. These clones, from mid ripening stage, were expressed in a heterologous system in tobacco plants by agroinfiltration. The PH activity was detected using the Feigl-Anger method and quantifying the hydrogen cyanide released with prunasin as substrate. Furthermore, ß-glucosidase activity was detected by Fast Blue BB salt and Umbelliferyl method. Differences at the sequence level (SNPs) and in the activity assays were detected, although no correlation with bitterness was found.

Cyanogenic Glucosides and Derivatives in Almond and Sweet Cherry Flower Buds from Dormancy to Flowering.

Almond and sweet cherry are two economically important species of the Prunus genus. They both produce the cyanogenic glucosides prunasin and amygdalin. As part of a two-component defense system, prunasin and amygdalin release toxic hydrogen cyanide upon cell disruption. In this study, we investigated the potential role within prunasin and amygdalin and some of its derivatives in endodormancy release of these two Prunus species. The content of prunasin and of endogenous prunasin turnover products in the course of flower development was examined in five almond cultivars - differing from very early to extra-late in flowering time - and in one sweet early cherry cultivar. In all cultivars, prunasin began to accumulate in the flower buds shortly after dormancy release and the levels dropped again just before flowering time. In almond and sweet cherry, the turnover of prunasin coincided with increased levels of prunasin amide whereas prunasin anitrile pentoside and ß-D-glucose-1-benzoate were abundant in almond and cherry flower buds at certain developmental stages. These findings indicate a role for the turnover of cyanogenic glucosides in controlling flower development in Prunus species.

Gene Expression Analysis of Plum pox virus (Sharka) Susceptibility/Resistance in Apricot (Prunus armeniaca L.).

RNA-Seq has proven to be a very powerful tool in the analysis of the Plum pox virus (PPV, sharka disease)/Prunus interaction. This technique is an important complementary tool to other means of studying genomics. In this work an analysis of gene expression of resistance/susceptibility to PPV in apricot is performed. RNA-Seq has been applied to analyse the gene expression changes induced by PPV infection in leaves from two full-sib apricot genotypes, "Rojo Pasión" and "Z506-7", resistant and susceptible to PPV, respectively. Transcriptomic analyses revealed the existence of more than 2,000 genes related to the pathogen response and resistance to PPV in apricot. These results showed that the response to infection by the virus in the susceptible genotype is associated with an induction of genes involved in pathogen resistance such as the allene oxide synthase, S-adenosylmethionine synthetase 2 and the major MLP-like protein 423. Over-expression of the Dicer protein 2a may indicate the suppression of a gene silencing mechanism of the plant by PPV HCPro and P1 PPV proteins. On the other hand, there were 164 genes involved in resistance mechanisms that have been identified in apricot, 49 of which are located in the PPVres region (scaffold 1 positions from 8,050,804 to 8,244,925), which is responsible for PPV resistance in apricot. Among these genes in apricot there are several MATH domain-containing genes, although other genes inside (Pleiotropic drug resistance 9 gene) or outside (CAP, Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis-related 1 protein; and LEA, Late embryogenesis abundant protein) PPVres region could also be involved in the resistance.

Recent advancements to study flowering time in almond and other Prunus species.

Flowering time is an important agronomic trait in almond since it is decisive to avoid the late frosts that affect production in early flowering cultivars. Evaluation of this complex trait is a long process because of the prolonged juvenile period of trees and the influence of environmental conditions affecting gene expression year by year. Consequently, flowering time has to be studied for several years to have statistical significant results. This trait is the result of the interaction between chilling and heat requirements. Flowering time is a polygenic trait with high heritability, although a major gene Late blooming (Lb) was described in "Tardy Nonpareil." Molecular studies at DNA level confirmed this polygenic nature identifying several genome regions (Quantitative Trait Loci, QTL) involved. Studies about regulation of gene expression are scarcer although several transcription factors have been described as responsible for flowering time. From the metabolomic point of view, the integrated analysis of the mechanisms of accumulation of cyanogenic glucosides and flowering regulation through transcription factors open new possibilities in the analysis of this complex trait in almond and in other Prunus species (apricot, cherry, peach, plum). New opportunities are arising from the integration of recent advancements including phenotypic, genetic, genomic, transcriptomic, and metabolomics studies from the beginning of dormancy until flowering.

Determinación d compuestos cianogénicos amigdaina y prunasina en semillas de almendras (prunus dulcis l) utilizando cromatografía liquida de alta resolución / Determination of cyanogenic compound amygdalin and prunasin in alond kernels (prrunus dulcis l) by using liquid chromatography / Detrminação de ccompostos cianogênicos amigdalina eprunasina em seementes e amêndoa (prunus dulcis miller) cromatografia líquida de alta resolução

El objetivo del presente trabajo fue aplicar un técnica para determinar y cuantificar por separado los compuestos cianogénicos que pueden estar presentes en la semilla de almendra madura (Prunus dulcis). Entre los métodos encontrados, se seleccionó la cromatografía de líquidos de alta resolución (HPLC), que permite la cuantificación de los glucósidos cianogénicos amigdalina y prunasina por separado, adecuando diferentes procedimientos de extracción como el tamaño de partículas que influye en el proceso de liofilización, donde a menor superficie mayor área de contacto para la sublimación. Se ensayaron muestras sin grasa y con grasa, utilizando los resultados con muestras con grasa, dados los resultados obtenidos. Se utilizó metanol 100% como extractante de los glucósidos cianogénicos, resultando una concentración de amigdalina máxima a partir un tiempo de extracción de 12 horas y como fase móvil acetonitrilo/agua (20:80), se obtiene amigdalina, con una concentración de 9,8 mg/100g de muestra seca. Los cromatogramas obtenidos presentan tiempo de retención (Tr), Amigdalina: 3,4 y Prunasina, 5,7, dos picos con excelente resolución, por lo tanto las condiciones anteriores se pueden utilizar para la identificación y cuantificación de amigdalina y prunasina.

The aim of this study was to apply a technique to identify and quantify separately also cyanogenic compounds that may be present in the mature seed almond (Prunus dulcis). Among the methods selected the chromatography of liquids of high resolution (HPLC), that permit the quantification of the glycosides for the separation process of Freeze Dry where there is less surface there is more contact to sublimation without fat samples, looking at the obtain results and supported by other investigations, the use of 100 % methanol extract as a mobile phase acetonitrile-water (80:20) the results obtained of the glycosides cyanogenics resulting in a concentration of maximum amygdalin from the time of extraction of twelve hours, amygdalin is obtained, with a concentration of 9,8 mg / 100 g of dry sample. The chromatograms obtained a time of retention (Tr), amygdalin 3,4 and prunasin 5,7 two peaks with excellent resolution, to the above conditions can be used for analysis by HPLC, identification and quantification of amygdalin and prunasina.

Neste trabalho, a técnica é aplicada para determinar e também para quantificar separadamente compostos cianogénicos que podem estar presentes na semente madura amêndoa (Prunus dulcis). Métodos encontrados é seleccionado de cromatografia líquida de alta eficiência (HPLC), que permite a quantificação dos glicosídeos separar adaptar diferentes técnicas de extracção, tais como o tamanho de partícula influencia o processo de liofilização, onde a área de superfície maior menor sublimação contacto com desengradas amostras de gordura e usando os resultados com amostras desengorduradas, Tendo em vista os resultados obtidos, e suportados por outras pesquisas metanol a 100 % foi usado como o agente de extracção e como fase móvel acetonitrilo/água (80:20) de glicósidos cianogénicos, resultando numa concentração elevada de amigdalina a partir de um tempo de extracção de 12 horas. Amigdalina é obtido, com uma concentração de 9,8 mg / 100 g de amostra seca. Os cromatogramas apresentados tempo de retenção (Tr), Amygdalin: 3,4 e prunasina 5,7 dois picos com excelente resolução, com as condições acima podem ser utilizados para a análise por HPLC. identificação e quantificação de amigdalina e prunasina.

Determinación de compuestos cianogénicos amigdalina y prunasina en semillas de almendras (prunus dulcis l) utilizando cromatografía liquida de alta resolución / Determination of cyanogenic compound amygdalin and prunasin in almond kernels (prunus dulcis l) by using liquid chromatography / Determinação de compostos cianogênicos amigdalina e prunasina em sementes e amêndoa (prunus dulcis miller) cromatografia líquida de alta resolução

El objetivo del presente trabajo fue aplicar un técnica para determinar y cuantificar por separado los compuestos cianogénicos que pueden estar presentes en la semilla de almendra madura (Prunus dulcis). Entre los métodos encontrados, se seleccionó la cromatografía de líquidos de alta resolución (HPLC), que permite la cuantificación de los glucósidos cianogénicos amigdalina y prunasina por separado, adecuando diferentes procedimientos de extracción como el tamaño de partículas que influye en el proceso de liofilización, donde a menor superficie mayor área de contacto para la sublimación. Se ensayaron muestras sin grasa y con grasa, utilizando los resultados con muestras con grasa, dados los resultados obtenidos. Se utilizó metanol 100% como extractante de los glucósidos cianogénicos, resultando una concentración de amigdalina máxima a partir un tiempo de extracción de 12 horas y como fase móvil acetonitrilo/agua (20:80), se obtiene amigdalina, con una concentración de 9,8 mg/100g de muestra seca. Los cromatogramas obtenidos presentan tiempo de retención (Tr), Amigdalina: 3,4 y Prunasina, 5,7, dos picos con excelente resolución, por lo tanto las condiciones anteriores se pueden utilizar para la identificación y cuantificación de amigdalina y prunasina.

The aim of this study was to apply a technique to identify and quantify separately also cyanogenic compounds that may be present in the mature seed almond (Prunus dulcis). Among the methods selected the chromatography of liquids of high resolution (HPLC), that permit the quantification of the glycosides for the separation process of Freeze Dry where there is less surface there is more contact to sublimation without fat samples, looking at the obtain results and supported by other investigations, the use of 100 % methanol extract as a mobile phase acetonitrile-water (80:20) the results obtained of the glycosides cyanogenics resulting in a concentration of maximum amygdalin from the time of extraction of twelve hours, amygdalin is obtained, with a concentration of 9,8 mg / 100 g of dry sample. The chromatograms obtained a time of retention (Tr), amygdalin 3,4 and prunasin 5,7 two peaks with excellent resolution, to the above conditions can be used for analysis by HPLC, identification and quantification of amygdalin and prunasina.

Neste trabalho, a técnica é aplicada para determinar e também para quantificar separadamente compostos cianogénicos que podem estar presentes na semente madura amêndoa (Prunus dulcis). Métodos encontrados é seleccionado de cromatografia líquida de alta eficiência (HPLC), que permite a quantificação dos glicosídeos separar adaptar diferentes técnicas de extracção, tais como o tamanho de partícula influencia o processo de liofilização, onde a área de superfície maior menor sublimação contacto com desengradas amostras de gordura e usando os resultados com amostras desengorduradas, Tendo em vista os resultados obtidos, e suportados por outras pesquisas metanol a 100 % foi usado como o agente de extracção e como fase móvel acetonitrilo/água (80:20) de glicósidos cianogénicos, resultando numa concentração elevada de amigdalina a partir de um tempo de extracção de 12 horas. Amigdalina é obtido, com uma concentração de 9,8 mg / 100 g de amostra seca. Os cromatogramas apresentados tempo de retenção (Tr), Amygdalin: 3,4 e prunasina 5,7 dois picos com excelente resolução, com as condições acima podem ser utilizados para a análise por HPLC. identificação e quantificação de amigdalina e prunasina.

Comparative genomics analysis in Prunoideae to identify biologically relevant polymorphisms.

Prunus is an economically important genus with a wide range of physiological and biological variability. Using the peach genome as a reference, sequencing reads from four almond accessions and one sweet cherry cultivar were used for comparative analysis of these three Prunus species. Reference mapping enabled the identification of many biological relevant polymorphisms within the individuals. Examining the depth of the polymorphisms and the overall scaffold coverage, we identified many potentially interesting regions including hundreds of small scaffolds with no coverage from any individual. Non-sense mutations account for about 70 000 of the 13 million identified single nucleotide polymorphisms (SNPs). Blast2GO analyses on these non-sense SNPs revealed several interesting results. First, non-sense SNPs were not evenly distributed across all gene ontology terms. Specifically, in comparison with peach, sweet cherry is found to have non-sense SNPs in two 1-aminocyclopropane-1-carboxylate synthase (ACS) genes and two 1-aminocyclopropane-1-carboxylate oxidase (ACO) genes. These polymorphisms may be at the root of the nonclimacteric ripening of sweet cherry. A set of candidate genes associated with bitterness in almond were identified by comparing sweet and bitter almond sequences. To the best of our knowledge, this is the first report in plants of non-sense SNP abundance in a genus being linked to specific GO terms.

Prunasin hydrolases during fruit development in sweet and bitter almonds.

Amygdalin is a cyanogenic diglucoside and constitutes the bitter component in bitter almond (Prunus dulcis). Amygdalin concentration increases in the course of fruit formation. The monoglucoside prunasin is the precursor of amygdalin. Prunasin may be degraded to hydrogen cyanide, glucose, and benzaldehyde by the action of the ß-glucosidase prunasin hydrolase (PH) and mandelonitirile lyase or be glucosylated to form amygdalin. The tissue and cellular localization of PHs was determined during fruit development in two sweet and two bitter almond cultivars using a specific antibody toward PHs. Confocal studies on sections of tegument, nucellus, endosperm, and embryo showed that the localization of the PH proteins is dependent on the stage of fruit development, shifting between apoplast and symplast in opposite patterns in sweet and bitter cultivars. Two different PH genes, Ph691 and Ph692, have been identified in a sweet and a bitter almond cultivar. Both cDNAs are 86% identical on the nucleotide level, and their encoded proteins are 79% identical to each other. In addition, Ph691 and Ph692 display 92% and 86% nucleotide identity to Ph1 from black cherry (Prunus serotina). Both proteins were predicted to contain an amino-terminal signal peptide, with the size of 26 amino acid residues for PH691 and 22 residues for PH692. The PH activity and the localization of the respective proteins in vivo differ between cultivars. This implies that there might be different concentrations of prunasin available in the seed for amygdalin synthesis and that these differences may determine whether the mature almond develops into bitter or sweet.

Disruption of endosperm development: an inbreeding effect in almond (Prunus dulcis).

A homozygous self-compatible almond, originated from self-fertilization of a self-compatible genotype and producing a reasonable yield following open pollination, exhibited a very high fruit drop rate when self-pollinated. To investigate whether fruit dropping in this individual is related to an abnormal development of the embryo sac following self-fertilization, histological sections of ovaries from self and cross-pollinated flowers were observed by light microscopy. Additionally, the presence of pollen tubes in the ovary and fruit set were determined for both types of pollination. Despite pollen tubes reached the ovary after both pollinations, differences in embryo sac and endosperm development after fertilization were found. Thus, while for cross-fertilized ovules a pro-embryo and an endosperm with abundant nuclei were generally observed, most self-fertilized ovules remained in a previous developmental stage in which the embryo sac was not elongated and endosperm nuclei were absent. Although 30 days after pollination fruit set was similar for both pollination types, at 60 days it was significantly reduced for self-pollination. These results provide evidence that the high fruit drop in this genotype is the consequence of a disrupted development of the endosperm, what could be an expression of its high level of inbreeding.

Tissue and cellular localization of individual beta-glycosidases using a substrate-specific sugar reducing assay.

Traditional methods to localize beta-glycosidase activity in tissue sections have been based on incubation with the general substrate 6-bromo-2-naphthyl-beta-d-glucopyranoside. When hydrolysed in the presence of salt zinc compounds, 6-bromo-2-naphthyl-beta-d-glucopyranoside affords the formation of an insoluble coloured product. This technique does not distinguish between different beta-glycosidases present in the tissue. To be able to monitor the occurrence of individual beta-glycosidases in different tissues and cell types, we have developed a versatile histochemical method that can be used for localization of any beta-glycosidase that upon incubation with its specific substrate releases a reducing sugar. Experimentally, the method is based on hydrolysis of the specific substrate followed by oxidation of the sugar released by a tetrazolium salt (2,3,5-triphenyltetrazolium chloride) that forms a red insoluble product when reduced. The applicability of the method was demonstrated by tissue and cellular localization of two beta-glucosidases, amygdalin hydrolase and prunasin hydrolase, in different tissues and cell types of almond. In those cases where the analysed tissue had a high content of reducing sugars, this resulted in strong staining of the background. This interfering staining of the background was avoided by prior incubation with sodium borohydride. The specificity of the devised method was demonstrated in a parallel localization study using a specific antibody towards prunasin hydrolase.

Bitterness in almonds.

Bitterness in almond (Prunus dulcis) is determined by the content of the cyanogenic diglucoside amygdalin. The ability to synthesize and degrade prunasin and amygdalin in the almond kernel was studied throughout the growth season using four different genotypes for bitterness. Liquid chromatography-mass spectrometry analyses showed a specific developmentally dependent accumulation of prunasin in the tegument of the bitter genotype. The prunasin level decreased concomitant with the initiation of amygdalin accumulation in the cotyledons of the bitter genotype. By administration of radiolabeled phenylalanine, the tegument was identified as a specific site of synthesis of prunasin in all four genotypes. A major difference between sweet and bitter genotypes was observed upon staining of thin sections of teguments and cotyledons for beta-glucosidase activity using Fast Blue BB salt. In the sweet genotype, the inner epidermis in the tegument facing the nucellus was rich in cytoplasmic and vacuolar localized beta-glucosidase activity, whereas in the bitter cultivar, the beta-glucosidase activity in this cell layer was low. These combined data show that in the bitter genotype, prunasin synthesized in the tegument is transported into the cotyledon via the transfer cells and converted into amygdalin in the developing almond seed, whereas in the sweet genotype, amygdalin formation is prevented because the prunasin is degraded upon passage of the beta-glucosidase-rich cell layer in the inner epidermis of the tegument. The prunasin turnover may offer a buffer supply of ammonia, aspartic acid, and asparagine enabling the plants to balance the supply of nitrogen to the developing cotyledons.