Stefan Jouannic

Analysis of expressed sequence tags from oil palm (Elaeis guineensis)

This is the first report of a systematic study of genes expressed by means of expressed sequence tag (EST) analysis in oil palm, a species of the Arecales order, a phylogenetically key clade of monocotyledons that is not widely represented in the sequence databases. Five different cDNA libraries were generated from male and female inflorescences, shoot apices and zygotic embryos and unidirectional systematic sequencing was performed. A total of 2411 valid EST sequences were thus obtained. Cluster analysis enabled the identification of 209 groups of related sequences and 1874 singletons. Putative functions were assigned to 1252 of the set of 2083 non‐redundant ESTs obtained. The EST database described here is a first step towards gene discovery and cDNA array‐based expression analysis in oil palm.

Reproductive developmental complexity in the African oil palm (Elaeis guineensis, Arecaceae)

Species of the palm family (Arecaceae) are remarkably diverse in their inflorescence and floral morphologies, which make them a particularly interesting group for studies of reproductive development and its evolution. Using light and scanning electron microscopy, we describe inflorescence and flower development in the African oil palm Elaeis guineensis from the initiation of the inflorescence meristem to flower maturity. In mature palms, the inflorescence develops over 2-3 years and is characterized by individual stages within which differentiation may be either relatively slow, as in the case of early inflorescence meristem development, or rapid, as in the case of flower organogenesis. The female inflorescence bears floral triads composed of single pistillate flowers flanked by two abortive staminate flowers, whereas the male inflorescence contains single functional staminate flowers. This suggests a possible evolutionary movement from an ancestral hermaphrodite inflorescence form containing fully functional floral triads to the situation of temporal dioecy observed at present. Wild type flowers are compared to those bearing an epigenetic homeotic abnormality, known as mantled, involving an alteration of the identity of the organs in the fertile and sterile androecium.

An unusual protein kinase displaying characteristics of both the serine/threonine and tyrosine families is encoded by the Arabidopsis thaliana gene ATN1☆

Abstract A cDNA clone, ATN1 , coding for a novel protein kinase, was isolated from an Arabidopsis thaliana inflorescence cDNA library. The deduced ATN1 protein sequence of 356 amino acid residues contains all the invariant hallmarks of the protein kinase superfamily. The ATN1 protein is however unusual in that it contains not only amino acid motifs characteristic of the protein serine/threonine kinase family, but also residues typical of protein tyrosine kinases. The ATN1 protein is not closely related to any other protein kinases characterised to date in either plants or other organisms. A database search revealed only 6 characterised protein kinases which shared greater than 30% identities with ATN1 (ranging from 33.5–41.4%) in their catalytic domains; each of the related sequences has mixed serine/threonine and tyrosine kinase affinities. The N-terminal region of ATN1 displays and N -myristoylation motif similar to those found in a number of other protein kinases. Although ATN1 does not belong to an established plant protein kinase subfamily, it is evolutionarily closest to a group which includes kinases involved in transmembrane signalling (the ‘receptor-like kinases’). The ATN1 gene forms part of a small multigene family and is expressed in all organs of the plant studied.

Plant MAP kinase kinase kinases structure, classification and evolution

The increasing number of reports describing plant MAP kinase signalling components reflects the cardinal role that MAP kinase pathways are likely to play during plant growth and development. Relationship and structural analyses of plant MAP kinase kinase kinase related cDNAs and genes established, on one hand, the PMEKKs, which may be distinguished into the alpha, beta, gamma, and zeta groups, and, on the other hand, the PRAFs that consist of the delta, eta and theta groups. Plant MAP3Ks are characterized by different primary structures, but conserved within a single group. A relationship analysis, which included animal, fungal and plant MAP3Ks, revealed a high degree of diversity among this biochemically established set of proteins, thus suggesting a range of biological functions. Four major families emerged, namely the MEKK/STE11, including the PMEKKs, the RAF, including the PRAFs, as well as the MLK and CDC7 families. These four families showed phylum-dependent distributions. Signature sequences characterizing the RAF family and the RAF subfamilies have been evidenced. However, no equivalent sequence motifs were identified for the MEKK/STE11 family, which is highly heterogeneous.

MADS box genes in oil palm (Elaeis guineensis): Patterns in the evolution of the SQUAMOSA, DEFICIENS, GLOBOSA, AGAMOUS, and SEPALLATA subfamilies

MADS box genes code for a large family of transcription factors which regulate development in higher plants, notably flower formation. We describe here a study of members of the MADS box gene family in oil palm (Elaeis guineensis Jacq.), a representative of the family Arecaceae and order Arecales, a key group of monocotyledons which has been unreported in previous phylogenetic reconstructions of the different recognized clades of MADS box genes. In this study, 13 oil palm MADS box genes were identified and characterized. They were found to belong to five different subfamilies, namely, the previously defined SQUAMOSA, AGAMOUS, AGAMOUS-like2, DEFICIENS, and GLOBOSA groups. Genes belonging to each of these groups play a critical role in the determination of flower structure as defined by the ABCDE model. The in planta expression profiles of the oil palm MADS box genes were studied by RT-PCR and phylogenetic sequence diversity within individual subfamilies was investigated by comparing their deduced protein sequences with those of other angiosperms. Most of the oil palm sequences studied were observed to group with distinct supported clades within their subfamily. Some unexpected groupings were observed between monocot sequences (including oil palm ones) of non-Poaceae origin, probably illustrating the importance of obtaining adequate taxon representation in monocot molecular phylogenies.

EgAP2-1, an AINTEGUMENTA-like (AIL) gene expressed in meristematic and proliferating tissues of embryos in oil palm

In order to better understand the developmental processes that govern the formation of somatic embryos in oil palm (Elaeis guineensis Jacq.), we investigated the transcription factor genes expressed during embryogenesis in this species. The AP2/EREBP transcription factor family includes the AP2 subgroup, which contains several proteins that play important roles in plant development. We identified and characterized EgAP2-1, which codes for a protein that contains two AP2 domains similar to those of the transcription factor BABYBOOM (BBM) and more generally AINTEGUMENTA-like (AIL) proteins of the AP2 subgroup. In a similar way to related genes from eudicots, ectopic expression of EgAP2-1 in transgenic Arabidopsis plants alters leaf morphology and enhances regeneration capacity. In oil palm, EgAP2-1 transcripts accumulate to the greatest extent in zygotic embryos. This expression pattern was investigated in more detail by in-situ hybridization, revealing that in both zygotic and somatic embryos, EgAP2-1 expression is concentrated in proliferating tissues associated with the early development of leaf primordia, root initials and provascular tissues.

Mitogen-activated protein kinases are developmentally regulated during stress-induced microspore embryogenesis in Brassica napus L

Plant mitogen-activated protein kinase (MAPK) cascades are involved in extracellular stress signalling pathways, leading to different cellular responses. Stress-induced microspore embryogenesis involves the internalization of an extracellular stress signal, generating a number of cellular responses where MAPK cascades might be involved. These responses include a change of the developmental programme, the entry into an early proliferative stage and, subsequently, into differentiation stages during haploid embryogenesis. In this work we studied the expression during microspore embryogenesis of several kinases, to assess their putative role in these events. The known Brassica napus MAP kinase kinase kinases (MAP3Ks BnMAP3Kα1, BnMAP3Kβ1 and BnMAP3Kε, the BnBSKθ kinase and B. napus extracellular signal-regulated kinase (ERK) homologues were analysed by electron microscope (EM) in situ hybridization, immuno-gold labelling, immunofluorescence and western blotting. The differential in situ expression of these kinases suggests a role for them during embryogenesis. Two different expression patterns were observed, indicating a different regulation. BnMAP3Kα1, BnMAP3Kε, and the ERKs showed a pattern consistent with a role mainly in proliferative events. Conversely, BnMAP3Kβ1 and BnBSKθ, presented a pattern that suggested an involvement in differentiation stages. In addition, ERK homologues migrate to the nucleus immediately after induction, being found in a phosphorylated state in a larger amount.

Characterisation of novel plant genes encoding MEKK/STE11 and RAF-related protein kinases

Various elements of the MAP kinase module have been isolated in plants. We describe here the characterisation of 14 new plant cDNAs and genes encoding putative MAP kinase kinase kinases (MAP3Ks) related to the MEKK/STE11 and RAF protein kinases. Plant MAP3Ks are characterised by a variety of primary structures conserved within closely related proteins. Southern blot analysis suggests that plant MAP3Ks are heterogenous in their genomic structure, existing either as single copy genes or as small gene families. An RT-PCR analysis showed that in Arabidopsis thaliana, all organs studied contain detectable levels of transcripts of each of the MAP3K genes identified; however, signals obtained with mature pollen were weak or non-existent except for AtMAP3Kgamma. None of the reported genes share a cell-cycle or a cold stress regulated expression.

Divergent Expression Patterns of miR164 and CUP-SHAPED COTYLEDON Genes in Palms and Other Monocots: Implication for the Evolution of Meristem Function in Angiosperms

In order to understand how the morphology of plant species has diversified over time, it is necessary to decipher how the underlying developmental programs have evolved. The regulatory network controlling shoot meristem activity is likely to have played an important role in morphological diversification and useful insights can be gained by comparing monocots and eudicots. These two distinct monophyletic groups of angiosperms diverged 130 Ma and are characterized by important differences in their morphology. Several studies of eudicot species have revealed a conserved role for NAM and CUC3 genes in meristem functioning and pattern formation through the definition of morphogenetic boundaries during development. In this study, we show that NAM- and CUC3-related genes are conserved in palms and grasses, their diversification having predated the radiation of monocots and eudicots. Moreover, the NAM-miR164 posttranscriptional regulatory module is also conserved in palm species. However, in contrast to the CUC3-related genes, which share a similar expression pattern between the two angiosperm groups, the expression domain of the NAM-miR164 module differs between monocot and eudicot species. In our studies of spatial expression patterns, we compared existing eudicot data with novel results from our work using two palm species (date palm and oil palm) and two members of the Poaceae (rice and millet). In addition to contrasting results obtained at the gene expression level, major differences were also observed between eudicot and monocot NAM-related genes in the occurrence of putative cis-regulatory elements in their promoter sequences. Overall, our results suggest that although NAM- and CUC3-related proteins are functionally equivalent between monocots and eudicots, evolutionary radiation has resulted in heterotopy through alterations in the expression domain of the NAM-miR164 regulatory module.

The shoot apical meristem of oil palm (Elaeis guineensis; Arecaceae): developmental progression and dynamics.

BACKGROUND AND AIMS Oil palm, an unbranched perennial monocotyledon, possesses a single shoot apical meristem (SAM), which is responsible for the initiation of the entire above-ground structure of the plant. To compare the palm SAM structure with those of other monocots and to study variations in its structure throughout the life of the plant, its organization was characterized from the embryonic stage to that of the reproductive plant. METHODS SAM structure was studied by a combination of stained histological sections, light and confocal microscopy, and serial section-based three-dimensional reconstructions. KEY RESULTS The oil palm SAM is characterized by two developmental phases: a juvenile phase with a single tunica-corpus structure displaying a gradual increase in size; and a mature phase characterized by a stable size, a modified shape and an established histological zonation pattern. In mature plants, fluctuations in SAM shape and volume occur, mainly as a consequence of changes in the central zone, possibly in relation to leaf initiation. CONCLUSIONS Development of the oil palm SAM is characterized by a juvenile to mature phase transition accompanied by establishment of a zonal pattern and modified shape. SAM zonation is dynamic during the plastochron period and displays distinct features compared with other monocots.