Setting up Agrobacterium tumefaciens-mediated transformation of the tropical legume Aeschynomene evenia, a powerful tool for studying gene function in Nod Factor-independent symbiosis.

Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene evenia has the remarkable property of being nodulated by photosynthetic Rhizobia without the intervention of Nod Factors (NodF). Thereby, A. evenia has emerged as a working model for investigating the NodF-independent symbiosis. Despite the availability of numerous resources and tools to study the molecular basis of this atypical symbiosis, the lack of a transformation system based on Agrobacterium tumefaciens significantly limits the range of functional approaches. In this report, we present the development of a stable genetic transformation procedure for A. evenia. We first assessed its regeneration capability and found that a combination of two growth regulators, NAA (= Naphthalene Acetic Acid) and BAP (= 6-BenzylAminoPurine) allows the induction of budding calli from epicotyls, hypocotyls and cotyledons with a high efficiency in media containing 0,5 µM NAA (up to 100% of calli with continuous stem proliferation). To optimize the generation of transgenic lines, we employed A. tumefaciens strain EHA105 harboring a binary vector carrying the hygromycin resistance gene and the mCherry fluorescent marker. Epicotyls and hypocotyls were used as the starting material for this process. We have found that one growth medium containing a combination of NAA (0,5 µM) and BAP (2,2 µM) was sufficient to induce callogenesis and A. tumefaciens strain EHA105 was sufficiently virulent to yield a high number of transformed calli. This simple and efficient method constitutes a valuable tool that will greatly facilitate the functional studies in NodF-independent symbiosis.

Origins and insights into the historic Judean date palm based on genetic analysis of germinated ancient seeds and morphometric studies.

Germination of 2000-year-old seeds of Phoenix dactylifera from Judean desert archaeological sites provides a unique opportunity to study the Judean date palm, described in antiquity for the quality, size, and medicinal properties of its fruit, but lost for centuries. Microsatellite genotyping of germinated seeds indicates that exchanges of genetic material occurred between the Middle East (eastern) and North Africa (western) date palm gene pools, with older seeds exhibiting a more eastern nuclear genome on a gradient from east to west of genetic contributions. Ancient seeds were significantly longer and wider than modern varieties, supporting historical records of the large size of the Judean date. These findings, in accord with the region's location between east and west date palm gene pools, suggest that sophisticated agricultural practices may have contributed to the Judean date's historical reputation. Given its exceptional storage potentialities, the date palm is a remarkable model for seed longevity research.

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

Since the publication of this paper, it has become apparent that an error was made in the legend to Fig. 3 and the colours referring to occidental and oriental are the wrong way round. The authors apologise for this error, and a correct version of the legend to Fig. 3 is given below.

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.

In silico mining of microsatellites in coding sequences of the date palm (Arecaceae) genome, characterization, and transferability.

PREMISE OF THE STUDY: To complement existing sets of primarily dinucleotide microsatellite loci from noncoding sequences of date palm, we developed primers for tri- and hexanucleotide microsatellite loci identified within genes. Due to their conserved genomic locations, the primers should be useful in other palm taxa, and their utility was tested in seven other Phoenix species and in Chamaerops, Livistona, and Hyphaene. • METHODS AND RESULTS: Tandem repeat motifs of 3-6 bp were searched using a simple sequence repeat (SSR)-pipeline package in coding portions of the date palm draft genome sequence. Fifteen loci produced highly consistent amplification, intraspecific polymorphisms, and stepwise mutation patterns. • CONCLUSIONS: These microsatellite loci showed sufficient levels of variability and transferability to make them useful for population genetic, selection signature, and interspecific gene flow studies in Phoenix and other Coryphoideae genera.

Male-specific DNA markers provide genetic evidence of an XY chromosome system, a recombination arrest and allow the tracing of paternal lineages in date palm.

Whether sex chromosomes are differentiated is an important aspect of our knowledge of dioecious plants, such as date palm (Phoenix dactylifera). In this crop plant, the female individuals produce dates, and are thus the more valuable sex. However, there is no way to identify the sex of date palm plants before reproductive age, and the sex-determining mechanism is still unclear. To identify sex-linked microsatellite markers, we surveyed a set of 52 male and 55 female genotypes representing the geographical diversity of the species. We found three genetically linked loci that are heterozygous only in males. Male-specific alleles allowed us to identify the gender in 100% of individuals. These results confirm the existence of an XY chromosomal system with a nonrecombining XY-like region in the date palm genome. The distribution of Y haplotypes in western and eastern haplogroups allowed us to trace two male ancestral paternal lineages that account for all known Y diversity in date palm. The very low diversity associated with Y haplotypes is consistent with clonal paternal transmission of a nonrecombining male-determining region. Our results establish the date palm as a biological model with one of the most ancient sex chromosomes in flowering plants.

Characterization, high-resolution mapping and differential expression of three homologous PAL genes in Coffea canephora Pierre (Rubiaceae).

Phenylalanine ammonia lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway producing phenolics, widespread constituents of plant foods and beverages, including chlorogenic acids, polyphenols found at remarkably high levels in the coffee bean and long recognized as powerful antioxidants. To date, whereas PAL is generally encoded by a small gene family, only one gene has been characterized in Coffea canephora (CcPAL1), an economically important species of cultivated coffee. In this study, a molecular- and bioinformatic-based search for CcPAL1 paralogues resulted successfully in identifying two additional genes, CcPAL2 and CcPAL3, presenting similar genomic structures and encoding proteins with close sequences. Genetic mapping helped position each gene in three different coffee linkage groups, CcPAL2 in particular, located in a coffee genome linkage group (F) which is syntenic to a region of Tomato Chromosome 9 containing a PAL gene. These results, combined with a phylogenetic study, strongly suggest that CcPAL2 may be the ancestral gene of C. canephora. A quantitative gene expression analysis was also conducted in coffee tissues, showing that all genes are transcriptionally active, but they present distinct expression levels and patterns. We discovered that CcPAL2 transcripts appeared predominantly in flower, fruit pericarp and vegetative/lignifying tissues like roots and branches, whereas CcPAL1 and CcPAL3 were highly expressed in immature fruit. This is the first comprehensive study dedicated to PAL gene family characterization in coffee, allowing us to advance functional studies which are indispensable to learning to decipher what role this family plays in channeling the metabolism of coffee phenylpropanoids.

Phylogenetic utility of the nuclear genes AGAMOUS 1 and PHYTOCHROME B in palms (Arecaceae): an example within Bactridinae.

BACKGROUND AND AIMS: Molecular phylogenetic studies of palms (Arecaceae) have not yet provided a fully resolved phylogeny of the family. There is a need to increase the current set of markers to resolve difficult groups such as the Neotropical subtribe Bactridinae (Arecoideae: Cocoseae). We propose the use of two single-copy nuclear genes as valuable tools for palm phylogenetics. METHODS: New primers were developed for the amplification of the AGAMOUS 1 (AG1) and PHYTOCHROME B (PHYB) genes. For the AGAMOUS gene, the paralogue 1 of Elaeis guineensis (EgAG1) was targeted. The region amplified contained coding sequences between the MIKC K and C MADS-box domains. For the PHYB gene, exon 1 (partial sequence) was first amplified in palm species using published degenerate primers for Poaceae, and then specific palm primers were designed. The two gene portions were sequenced in 22 species of palms representing all genera of Bactridinae, with emphasis on Astrocaryum and Hexopetion, the status of the latter genus still being debated. KEY RESULTS: The new primers designed allow consistent amplification and high-quality sequencing within the palm family. The two loci studied produced more variability than chloroplast loci and equally or less variability than PRK, RPBII and ITS nuclear markers. The phylogenetic structure obtained with AG1 and PHYB genes provides new insights into intergeneric relationships within the Bactridinae and the intrageneric structure of Astrocaryum. The Hexopetion clade was recovered as monophyletic with both markers and was weakly supported as sister to Astrocaryum sensu stricto in the combined analysis. The rare Astrocaryum minus formed a species complex with Astrocaryum gynacanthum. Moreover, both AG1 and PHYB contain a microsatellite that could have further uses in species delimitation and population genetics. CONCLUSIONS: AG1 and PHYB provide additional phylogenetic information within the palm family, and should prove useful in combination with other genes to improve the resolution of palm phylogenies.

Cell cycle arrest characterizes the transition from a bisexual floral bud to a unisexual flower in Phoenix dactylifera.

BACKGROUND AND AIMS: Phoenix dactylifera (date palm) is a dioecious species displaying strong dimorphism between pistillate and staminate flowers. The mechanisms involved in the development of unisexual flowers are as yet unknown. METHODS: This paper describes the results of inflorescence and flower development studies using different histological and molecular cytological approaches. Nuclear integrity and cell division patterns in reproductive organs were investigated through DAPI staining and in situ hybridization using a histone H4 gene probe. KEY RESULTS: The earliest sex-related difference in flower buds is observed at an otherwise 'bisexual' stage, at which the number of cells in the gynoecium of pistillate flowers is higher than in their staminate counterparts. In the pistillate flower, staminodes (sterile stamens) display precocious arrest of development followed by cell differentiation. In the staminate flower, pistillodes (sterile gynoecium) undergo some degree of differentiation and their development ceases shortly after the ovule has been initiated. Staminode and pistillode cells exhibit nuclear integrity although they did not show any accumulation of histone H4 gene transcripts. CONCLUSIONS: These results strongly suggest that the developmental arrest of sterile sex organs and the subsequent unisexuality of date palm flowers result from a cessation of cell division and precocious cell differentiation rather than from cell death.

Contrasting globulin and cysteine proteinase gene expression patterns reveal fundamental developmental differences between zygotic and somatic embryos of oil palm.

Oil palm (Elaeis guineensis Jacq.) somatic embryos differ from zygotic embryos in that they accumulate only small amounts of storage proteins. We compared the balance between deposition and degradation of storage proteins during zygotic or somatic embryogenesis and germinative growth in the two types of embryos. During mid to late zygotic embryogenesis, storage proteins accumulated and globulin 7S (GLO7A) gene transcripts were detected, whereas neither protease activity nor cysteine proteinase (CPR) gene transcripts were detected. Globulin degradation occurred after 8 days of in vitro germination in zygotic embryos and was accompanied by a decrease in GLO7A transcripts. Transcripts of three cysteine proteinase genes of the papain family were detected as early as Day 2 of in vitro germination. Several proteolytically active protein bands were identified by zymography, and CPR-like proteins were detected with an antibody raised against the Vicia sativa L. cysteine proteinase CPR1. Protease activities and CPR-like proteins were observed from Day 8 onward when globulin degradation occurred. During somatic embryogenesis and subsequent germinative growth, only small amounts of storage proteins accumulated, even though GLO7A transcripts were detected. Two of the three cysteine proteinase genes were expressed throughout both somatic embryogenesis and germinative growth. Protease activities and CPR-like protein species were detected in somatic embryos at several developmental stages. In contrast to zygotic embryogenesis, the accumulation of globulins and their subsequent mobilization appear to be concomitant processes during somatic embryogenesis, which could explain the low accumulation of storage proteins in somatic embryos.

Functional characterization of two p-coumaroyl ester 3'-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis.

Chlorogenic acid (5-CQA) is one of the major soluble phenolic compounds that is accumulated in coffee green beans. With other hydroxycinnamoyl quinic acids (HQAs), this compound is accumulated in particular in green beans of the cultivated species Coffea canephora. Recent work has indicated that the biosynthesis of 5-CQA can be catalyzed by a cytochrome P450 enzyme, CYP98A3 from Arabidopsis. Two full-length cDNA clones (CYP98A35 and CYP98A36) that encode putative p-coumaroylester 3'-hydroxylases (C3'H) were isolated from C. canephora cDNA libraries. Recombinant protein expression in yeast showed that both metabolized p-coumaroyl shikimate at similar rates, but that only one hydroxylates the chlorogenic acid precursor p-coumaroyl quinate. CYP98A35 appears to be the first C3'H capable of metabolising p-coumaroyl quinate and p-coumaroyl shikimate with the same efficiency. We studied the expression patterns of both genes on 4-month old C. canephora plants and found higher transcript levels in young and in highly vascularized organs for both genes. Gene expression and HQA content seemed to be correlated in these organs. Histolocalization and immunolocalization studies revealed similar tissue localization for caffeoyl quinic acids and p-coumaroylester 3'-hydroxylases. The results indicated that HQA biosynthesis and accumulation occurred mainly in the shoot tip and in the phloem of the vascular bundles. The lack of correlation between gene expression and HQA content observed in some organs is discussed in terms of transport and accumulation mechanisms.

Tolerance of coffee (Coffea spp.) seeds to ultra-low temperature exposure in relation to calorimetric properties of tissue water, lipid composition, and cooling procedure.

The effect of exposure to ultra-low temperature (liquid nitrogen, LN) on viability of seeds desiccated to various water contents was investigated in 9 coffee species. Three groups of species could be distinguished based on seed survival after LN exposure. In group 1 species, no seedling production could be obtained after LN exposure due to endosperm injury. In group 2 species, recovery was very low or nil after rapid cooling, and only moderate after slow cooling. In group 3 species, very high percentages of seedling development were observed after both rapid and slow cooling. A high interspecific variability for the high moisture freezing limit was observed within the species of groups 2 and 3, since it ranged from 0.14 to 0.26 g H2O g-1 dry weight. A very highly significant correlation was found for those species between the unfreezable water content, as determined from DSC analysis, and the high moisture freezing limit of their seeds. No significant correlation was found between seed lipid content, which varied from 9.8 to 34.6% dry weight, and survival after LN exposure. However, a negative relationship was found between seed unfreezable water content and lipid content. Interspecific differences in fatty acid composition of seed lipids resulted in a high variability in the percentage of unsaturated fatty acids, which ranged from 28.7 to 54.4% among the 9 species studied. For all species studied, a highly significant correlation was found between the percentage of unsaturated fatty acids and the percentage of seedling recovery after rapid or slow cooling.

Variability in storage response within a coffee (Coffea spp.) core collection under slow growth conditions.

Anin vitro core collection of African coffee germplasm, structured in 32 diploid diversity groups, was established and conserved under slow growth for 3 years (6 subcultures). The initial objective was to store twenty accessions per group, with four replicates per accession. A statistical model was developed to analyse observations of survival rates within each diversity group. The goodness of fit of the model was shown. Survival analysis indicated a broad variability of the accessions in their response to the storage conditions and confirmed the importance of structuring the coffee complex down to the intraspecific level. Intra- and inter-group differences had consequences on the genetic representativity of thein vitro core collection. For practical purposes, conservation was carried on when the intra-group genetic drift was less than 50%.