Proteomic analysis of the development and germination of date palm (Phoenix dactylifera L.) zygotic embryos

By using a comparative proteomic approach (2‐DE coupled to MS/MS), the development, maturation, and germination of date palm zygotic embryos, have been studied. Proteins were trichloroacetic acid (TCA)–acetone–phenol extracted and resolved by 2‐DE in the 5–8 pH range. The total protein content and the number of spots resolved increased from early (12 weeks after pollination (WAP); 68.96 mg/g DW: 207 spots) to late (17 WAP; 240.85 mg/g DW: 261 spots) stages, decreasing upon germination (from 120.8 mg/g DW: 273 spots in mature embryos to 26.35 mg/g DW: 87 spots in 15 days after germination). Up to 194 spots showed qualitative or quantitative differences between stages. Statistical analysis of spot variation was performed by PCA, obtaining a more accurate grouping of the samples and determining the most discriminant spots. Samples were also clustered based on Pearson distance and Ward's minimum distance. Sixty‐five variable spots were subjected to MS analysis, resulting in 21 identifications. The identified proteins belong to the following functional categories: enzymes of glycolysis, tricarboxylic acid cycle, and carbohydrate biosynthesis, protein translation, storage (glutelin), and stress‐related proteins. The evolution pattern of the functional groups was examined and discussed in terms of metabolism adaptation to the different embryogenic and germination stages.     

A rapid and efficient method for the extraction of total DNA from mature leaves of the data palm (Phoenix dactylifera L.)

A method is presented for the rapid isolation of high-molecular-weight DNA from mature leaves of date palm (Phoenix dactylifera L.), using a CTAB-based buffer. The method yields up to 800 μg of DNA from 1 g of leaf tissues. The procedure was also suitable for DNA extraction from callus or buds from tissue culture. The DNA obtained through this method was a good substrate for at least seventeen restriction endonucleases. This method was also used to extract DNA from mature leaves of coconut and may be applicable to other species of palms.     

Microsatellite markers reveal high genetic diversity in date palm (<Emphasis Type="Italic">Phoenix dactylifera</Emphasis> L.) germplasm from Sudan

Genetic diversity in date palm germplasm from Sudan representing 37 female and 23 male accessions was investigated using 16 loci of microsatellite (SSR) primers. Eight female accessions from Morocco were included as reference material. The tested SSR markers showed a high level of polymorphism. A total of 343 alleles were detected at the 16 loci. The number of alleles per marker ranged from 14 to 44 with an average of 21.4 per locus. A high level of expected heterozygosity was observed among Sudan cultivars (0.841), Morocco cultivars (0.820) and male accessions (0.799). The results indicate that the genetic groups of the Sudan cultivars and/or males do not follow a clear geographic pattern. However, the morocco group showed significant differentiation in relation to the Sudan groups, as measured by F (ST) values and genetic distances. The effect of the methods of pollination and cultivar selection on the genetic structure was clearly detected by the weak clustering association that was observed for the majority of accessions originating from Sudan and Morocco as well. This suggests the need for further investigation on the genetic diversity of Sudanese date palm germplasm. A deeper insight will be revealed by a detailed analysis of populations originating from different geographic locations.     

Evaluation of extraction methods for the identification of proteins from date palm (Phoenix dactylifera L.) seed and flesh

Date fruits are well known to be very nutritious. Nevertheless, the protein contents of the fruit, particularly the seed and flesh, are still understudied, largely due to their difficult physical characteristics. This study was conducted to compare three different protein extraction methods which were the trichloroacetic acid (TCA)-acetone (TCA-A), phenol (Phe), and TCA-acetone-phenol (TCA-A-Phe), and to perform proteomic analysis on date palm seed and flesh. Phe extraction method showed the highest protein yields for both seed (8.26?mg/g) and flesh (1.57?mg/g). Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Phe, and TCA-A-Phe extraction methods were shown to be efficient in removing interfering compounds and gave well-resolved bands over a wide range of molecular weights. Following liquid chromatography–tandem mass spectrometry analysis, about 50–64% of extracted proteins were identified with known functions including those involved in glycolysis, Krebs cycle, defense, and storage. Phe protein extraction method was proven to be the optimal method for date flesh and seed.     

A neutral beta-D-glucan from dates of the date palm,Phoenix dactylifera L

Polysaccharides extracted from Libyan dates with hot water and 0.05 M NaOH were fractionated and purified by ion-exchange and gel-filtration chromatography. According to the methylation and hydrolysis analyses, the results indicate the D-glucan to be linear and to contain both (1-->3)- and (1-->4)-linkages. The anomeric NMR measurements confirm that the sugar residues are beta-glycosidically linked. This is the first report on the isolation of a neutral beta-D-glucan from dates.     

Comparative 2-DE proteomic analysis of date palm (Phoenix dactylifera L.) somatic and zygotic embryos

Two-dimensional gel electrophoresis coupled to mass spectrometry has been used to compare the proteome of date palm (Phoenix dactylifera L. cv. Deglet Nour) zygotic and somatic embryos. Proteins were trichloroacetic acid–acetone–phenol extracted, quantified, and resolved by 2-DE in the 5 to 8 pH range. Total protein content and number of resolved spots were higher in zygotic (110 ± 14.5 mg/g DW; 349 spots) than in somatic (70.96 ± 4.8 mg/g DW; 210 spots) embryos. The 2-DE map of both systems showed qualitative (263) and quantitative (72) differences. Statistical analysis of spot intensity was performed by PCA, obtaining two accurate groupings of the samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with average linkage algorithm of the Genesis software package. Sixty-three variable spots were subjected to mass spectrometry analysis, resulting in 23 identifications. Identified proteins were classified in the following functional categories; glycolysis (8 proteins), citrate cycle (1), ATP synthesis (1), carbohydrate biosynthesis (2), amino acids metabolism (1), stress related (4), storage (3), and with no function assigned for three of them. Most of the somatic embryo specific proteins identified belonged to glycolysis pathways, whereas those of the zygotic embryo to storage and stress-related proteins. Differences are discussed in terms of metabolism and biology of both types of embryos.     

An alkali-soluble heteroxylan from seeds of Phoenix dactylifera L

Alkali-soluble polysaccharides, isolated from the seeds of dates, have been investigated using methylation and partial hydrolysis studies. The polysaccharides are shown to contain D-xylose and 4-O-methyl-D-glucuronic acid in a molar ratio of 5:1. An aldobiouronic acid from hemicellulose was characterized, and investigation revealed that the hemicellulose consists of a polymer of (1-->4)-linked D-xylopranosyl residues having branches of D-xylopyranosyl and 4-O-methyl-alpha-D-glucopyranosyluronic acid.     

Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.     

Purification and characterization of membrane-bound peroxidase from date palm leaves (Phoenix dactylifera L.)

Peroxidase from date palm (Phoenix dactylifera L.) leaves was purified to homogeneity and characterized biochemically. The enzyme purification included homogenization, extraction of pigments followed by consecutive chromatographies on DEAE-Sepharose and Superdex 200. The purification factor for purified date palm peroxidase was 17 with 5.8% yield. The purity was checked by SDS and native PAGE, which showed a single prominent band. The molecular weight of the enzyme was approximately 55 kDa as estimated by SDS–PAGE. The enzyme was characterized for thermal and pH stability, and kinetic parameters were determined using guaiacol as substrate. The optimum activity was between pH 5–6. The enzyme showed maximum activity at 55 °C and was fairly stable up to 75 °C, with 42% loss of activity. Date palm leaves peroxidase showed K_m values of 0.77 and 0.045 mM for guaiacol and H₂O₂, respectively. These properties suggest that this enzyme could be a promising tool for applications in different analytical determinations as well as for treatment of industrial effluents at low cost.     

Molecular characterization of Mauritanian date palm cultivars using plasmid-like DNAs markers

Mauritanian date palm cultivars and progenies of two controlled crosses were analyzed according to the identity of mitochondrial plasmid-like DNAs. Starting from total genomic DNA and appropriate primers, polymerase chain reaction was designed to amplify either a 373-bp or a 265-bp fragments corresponding to the S and the R-plasmid respectively. Data proved that 5 cultivars out of 10 studied have exhibited the R-plasmid suggesting their resistance to the fusariosis. The existence of intra-cultivar variability has also been revealed in the cv. Ahmar. In addition, analysis throughout progenies of two controlled crosses suggested the strict maternal transmission of the date palms’ mitochondrial genome.     

Structure and biochemistry of endosperm breakdown in date palm (Phoenix dactylifera L.) seeds

Summary The zone of endosperm breakdown in the germinated date seed (Phoenix dactylifera L.) is a narrow area immediately adjacent to the surface of the enlarging cotyledon, or haustorium. The zone width is correlated with the amount of cell division in the adjacent region of the haustorium. The sequence of endosperm breakdown is: 1. protein bodies vacuolate, 2. storage cell walls become electron-transparent immediately adjacent to the protoplast of each endosperm cell, 3. all remaining cytoplasm and lipid bodies disappear, and 4. the remaining cell walls become electron-transparent and collapse against the haustorium surface. Two cell wall hydrolases are present—endo-mannanase (EC3.2.1.78) and -mannosidase (EC3.2.1.25). -mannosidase is detectable in the endosperm before germination. At germination, the major portion of activity is found in the softened endosperm. -mannanase is only detectable from germination and there is always hundreds of fold greater activity in the softened endosperm than elsewhere. Proteinase is detectable in trace amounts at germination in the softened endosperm but is also found in the haustorium at later stages. Isolated haustoria, incubated in extracted ivory nut (Phytelephas macrocarpa) mannan in buffer, cause no mannan breakdown. Haustoria, incubated in a solution of locust bean galactomannan, cause no decrease in galactomannan viscosity. Our observations suggest that although haustoria probably regulate mannan breakdown in the endosperm, they do not seem to secrete the hydrolytic enzymes concerned.     

The date palm (Phoenix dactylifera L.) micropropagation using completely mature female flowers

This study describes an efficient and reproducible protocol for in vitro date palm propagation using mature female flowers. It focuses on the promising proliferation capacity exhibited by a number of female flower tissues taken at the final developmental stage. This capacity resided in the ability to preserve minuscule zones in a juvenile state located at the floral organ armpits (sepals and petals). The originality of this method lies in the possibility of propagation of very rare varieties, particularly the genotypes that exist in only one copy without the excision of the plant mother, the source of the tissue collected to be cultivated, which was not the case for all previous methods. The findings revealed that 2,4-D at 1mg/l, most of the varieties tested showed reactivity. The success of this technique was also noted to depend on the concurrent control of various factors pertaining mainly to the hormonal composition of the culture medium and the appropriate time of tissue transfer, which depends on the proliferation state as well as the culture period. This study describes the nature of the proliferation from the mature female flowers and their outcome, particularly those at the origin of embryogenic and budding strains and discusses the advantages of this novel multiplication method as compared to the currently available ones.     

Staminodes evolution and in vitro development innovation in date palm (Phoenix dactylifera L.)

The in vitro cultivation of date palm staminodes (vestigial stamens) at different stages of female floral ontogenesis confirms the persistence at an immature state of such organs at all the floral differentiation stages. This is evidenced even in fully mature female flowers. Our study revealed the advanced developmental patterns of these rudimentary structures, which bear diverse morphogenetic potentialities. In vitro cultivation of staminodes provides new opportunities for in vitro regeneration of date palm. Such developmental processes were found to be modulated by the stage of floral differentiation, which closely reflected the level of staminode maturity. Development was also impacted by the composition and concentration in plant growth regulators (NAA, BAP and 2,4-D) of the culture media. The large morphogenetic plasticity of the staminodes disposed them to evolutionary variations of the date palm reproduction system. The practical benefits (micropropagation) and the fundamental interests (evolutionary process) of our investigation are discussed.     

Molecular evidence of hybridisation between the endemic Phoenix canariensis and the widespread P. dactylifera with Random Amplified Polymorphic DNA (RAPD) markers

We used RAPD markers to test whether morphologically intermediate individuals between the Canarian endemic Phoenix canariensis and the widespread P. dactylifera correspond to hybrids. Consistent with previous allozyme evidence, the scarcity of appropriate RAPD markers to distinguish P. dactylifera and P. canariensis indicated a close genetic relationship among these species. Only two of the 54 ten-mer primers (OPM-8 and OPK-14) tested in 221 individuals from 7 localities in different islands enabled us to unambiguously identify both species. While P. canariensis possesses two exclusive monomorphic bands of 1000 bp and 750 bp (for OPM-8 and OPM-14, respectively), dactylifera is characterised by two bands of 900 bp and 950 bp for the same primers. The additivity of these taxon-specific bands in the individuals that were morphologically intermediate provided, for the first time, firm evidence for their hybrid origin. Because these hybridisation capabilities pose clear threats to the survival of the endemic P. canariensis and some individuals that had been morphologically characterised as pure P. canariensis revealed later a hybrid nature in the RAPD analysis, we suggest that RAPD markers be used to estimate the possible incidence of introgression in the scarce extant natural populations of P. canariensis. This procedure will provide a straightforward means to select target populations to implement the in situ conservation strategies suggested previously on the basis of allozyme research.We thank Michel Ferry for providing samples of Phoenix dactylifera from the Hort del Gat Research Station on Date Palm and Arid Land Farming Systems in Elche (Spain). The authors wish to thank Jaime OShanahan for his wise comments about Phoenix canariensis. This research was funded by the Gobierno de Canarias (94-2614). A PhD Research Fellowship (Direccion General de Universidades, Gobierno de Canarias) to Gonzalez-Perez. is also acknowledged.     

Nuclear microsatellite markers for the date palm (Phoenix dactylifera L.): characterization and utility across the genus Phoenix and in other palm genera

A (GA)_n microsatellite‐enriched library was constructed and 16 nuclear simple sequence repeat (SSR) loci were characterized in Phoenix dactylifera. Across‐taxa amplification and genotyping tests showed the utility of most SSR markers in 11 other Phoenix species and the transferability of some of them in Elaeis guineensis, 11 species of Pritchardia, Pritchardiopsis jeanneneyi and six species of Astrocaryum. The first to be published for P. dactylifera, these new SSR resources are available for cultivar identification, pedigree analysis, germplasm diversity as well as genetic mapping studies.     

Histochemical and ultrastructural changes in the haustorium of date (Phoenix dactylifera L.)

Summary During imbibition ofPhoenix dactylifera embryos, all cotyledon cells show the same changes: protein and lipid bodies degrade, smooth endoplasmic reticulum (ER) increases in amount, and dictyosomes appear. At germination, the distal portion of the cotyledon expands to form the haustorium. At this time, epithelial cells have a dense cytoplasm with many extremely small vacuoles. Many ribosomes are present along with ER, dictyosomes, and mitochondria. The parenchyma cells have large vacuoles and a small amount of peripheral cytoplasm. Between 2 and 6 weeks after germination, epithelial cells still retain the dense cytoplasm and many organelles appear: glyoxysomes, large lipid bodies, amyloplasts, large osmiophilic bodies, and abundant rough and smooth ER which appear to merge into the plasmalemma. A thin electron-transparent inner wall layer with many small internal projections is added to the cell walls. Starch grains appear first in the subsurface and internal parenchyma and subsequently in the epithelium. Lipid bodies, glyoxysomes, protein, and osmiophilic bodies occur in the epithelial and subepithelial cell layers but not in the internal parenchyma. At 8 weeks after germination, the cytoplasm becomes electron transparent, vacuolation occurs, lipid bodies and osmiophilic bodies degrade, and the endomembranes disassemble. After 10 weeks, the cells are empty. These data support the hypothesis that the major functions of the haustorium are absorption and storage.     

Genetic structure of the date palm (Phoenix dactylifera) in the Old World reveals a strong differentiation between eastern and western populations

Background and Aims Date palms (Phoenix dactylifera, Arecaceae) are of great economic and ecological value to the oasis agriculture of arid and semi-arid areas. However, despite the availability of a large date palm germplasm spreading from the Atlantic shores to Southern Asia, improvement of the species is being hampered by a lack of information on global genetic diversity and population structure. In order to contribute to the varietal improvement of date palms and to provide new insights on the influence of geographic origins and human activity on the genetic structure of the date palm, this study analysed the diversity of the species.Methods Genetic diversity levels and population genetic structure were investigated through the genotyping of a collection of 295 date palm accessions ranging from Mauritania to Pakistan using a set of 18 simple sequence repeat (SSR) markers and a plastid minisatellite.Key Results Using a Bayesian clustering approach, the date palm genotypes can be structured into two different gene pools: the first, termed the Eastern pool, consists of accessions from Asia and Djibouti, whilst the second, termed the Western pool, consists of accessions from Africa. These results confirm the existence of two ancient gene pools that have contributed to the current date palm diversity. The presence of admixed genotypes is also noted, which points at gene flows between eastern and western origins, mostly from east to west, following a human-mediated diffusion of the species.Conclusions This study assesses the distribution and level of genetic diversity of accessible date palm resources, provides new insights on the geographic origins and genetic history of the cultivated component of this species, and confirms the existence of at least two domestication origins. Furthermore, the strong genetic structure clearly established here is a prerequisite for any breeding programme exploiting the effective polymorphism related to each gene pool.     

Methylation-sensitive amplification polymorphism in date palms (Phoenix dactylifera L.) and their off-shoots

DNA methylation plays an important role in the regulation of gene expression in eukaryotes. In this study, the extent and patterns of DNA methylation were assessed in date palm mother-plants and their off-shoots using the methylation-sensitive amplified polymorphism (MSAP) technique. Three types of bands were generated using 12 pairs of primers. Type I were present in both ECOR I + HPA II and ECOR I + MSP I lanes; type II were present in ECOR I + HPA II lanes, but not in ECOR I + MSP I lanes; and type III bands were present in ECOR I + MSP I lanes, but not in ECOR I + HPA II lanes. The total numbers of these three types of bands were 782, 55, and 34, respectively. Among these three types of bands, the polymorphic bands were, respectively, 37, 10, and 0. The distribution of polymorphic bands among mother-plants and off-shoots suggests the methylation variation was present in both the mother-plants and off-shoots. Forty- four out of these 47 polymorphic bands show clear difference between mother-plant and off-shoots: 38 were present only in off-shoots and 6 in both mother-plants and off-shoots. Compared to methylation status in mother-plants, the methylation variation during off-shoot growth of date palm can be characterized as a process involving primarily de-methylation. Hypomethylation of DNA in off-shoots, compared with mother-plants, reflects the marked expression of this molecular feature, which may be related to gene expression during off-shoot development. The methylation or de-methylation status of specific loci in the mother-plants and their off-shoots were probably random events.     

Characterization of ten date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia using AFLP and ISSR markers

Date palm is the most economically important plant in the Middle East due to its nutritionally valuable fruit. The development of accurate DNA fingerprints to characterize cultivars and the detection of genetic diversity are of great value for breeding programs. The present study explores the usefulness of ISSR and AFLP molecular markers to detect relationships among 10 date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia. Thirteen ISSR primers and six AFLP primer combinations were examined. The level of polymorphism among cultivars for ISSRs ranged from 20% to 100% with an average of 85%. Polymorphism levels for AFLPs ranged from 63% to 84% with an average of 76%. The total number of cultivar-specific markers was 241, 208 of which were generated from AFLP analysis. AJWA cultivar had the highest number of cultivar-specific ISSR markers, whereas DEK, PER, SUK-Q, SHA and MOS-H cultivars had the lowest. RAB and SHA cultivars had the most and least AFLP cultivar-specific markers, respectively. The highest pairwise similarity indices for ISSRs, AFLPs and combined markers were 84% between DEK (female) and PER (female), 81% between SUK-Q (male) and RAB (male), and 80% between SUK-Q (male) and RAB (male), respectively. The lowest similarity indices were 65% between TAB (female) and SUK-Q (male), 67% between SUK-A (female) and SUK-Q (male), and 67% between SUK-A (female) and SUK-Q (male). Cultivars of the same sex had higher pairwise similarities than those between cultivars of different sex. The Neighbor-Joining (NJ) tree generated from the ISSR dataset was not well resolved and bootstrap support for resolved nodes in the tree was low. AFLP and combined data generated completely resolved trees with high levels of bootstrap support. In conclusion, AFLP and ISSR approaches enabled discrimination among 10 date palm cultivars of from Saudi Arabia, which will provide valuable information for future improvement of this important crop.     

Phenolic compounds in date palm cultivars sensitive and resistant toFusarium oxysporum

In date palm (Phoenix dactylifera L.) leaves, the main compounds of the phenolic pool were quercetin and isorhamnetin heterosides, (+)-catechin and (-)-epicatechin. Although previously observed only in date palm fruits, 5-caffeoylshikimic acid (dactylifric acid) and its positional isomers (3-caffeoylshikimic acid and 4-caffeoylshikimic acid) were detected also in the leaves and roots. Quantitative, but not qualitative, differences between cultivars resistant and susceptible toFusarium oxysporum f. sp.albedinis during growth period were observed Acknowledgements: The authors are very grateful to Dr. Janati, Director of SCAS (INRA, Marrakech) for supplying the plant material used in this study. The study was funded by the Programme de Cooperation Franco-Marocaine (Al 473/90) between Cadi Ayyad University (Marrakech, Maroc) and Montpellier II University (France)