OsMADS26 Negatively Regulates Resistance to Pathogens and Drought Tolerance in Rice.

Functional analyses of MADS-box transcription factors in plants have unraveled their role in major developmental programs (e.g. flowering and floral organ identity) as well as stress-related developmental processes, such as abscission, fruit ripening, and senescence. Overexpression of the rice (Oryza sativa) MADS26 gene in rice has revealed a possible function related to stress response. Here, we show that OsMADS26-down-regulated plants exhibit enhanced resistance against two major rice pathogens: Magnaporthe oryzae and Xanthomonas oryzae. Despite this enhanced resistance to biotic stresses, OsMADS26-down-regulated plants also displayed enhanced tolerance to water deficit. These phenotypes were observed in both controlled and field conditions. Interestingly, alteration of OsMADS26 expression does not have a strong impact on plant development. Gene expression profiling revealed that a majority of genes misregulated in overexpresser and down-regulated OsMADS26 lines compared with control plants are associated to biotic or abiotic stress response. Altogether, our data indicate that OsMADS26 acts as an upstream regulator of stress-associated genes and thereby, a hub to modulate the response to various stresses in the rice plant.

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.

Micro-RNA-Regulated SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) Gene Expression and Cytokinin Accumulation Distinguish Early-Developing Male and Female Inflorescences in Oil Palm (Elaeis guineensis).

Sexual differentiation of inflorescences and flowers is important for reproduction and affects crop plant productivity. We report here on a molecular study of the process of sexual differentiation in the immature inflorescence of oil palm (Elaeis guineensis). This species is monoecious and exhibits gender diphasy, producing male and female inflorescences separately on the same plant in alternation. Three main approaches were used: small RNA-seq to characterise and study the expression of miRNA genes; RNA-seq to monitor mRNA accumulation patterns; hormone quantification to assess the role of cytokinins and auxins in inflorescence differentiation. Our study allowed the characterisation of 30 previously unreported palm MIRNA genes. In differential gene and miRNA expression studies, we identified a number of key developmental genes and miRNA-mRNA target modules previously described in relation to their developmental regulatory role in the cereal panicle, notably the miR156/529/535-SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) gene regulatory module. Gene enrichment analysis highlighted the importance of hormone-related genes, and this observation was corroborated by the detection of much higher levels of cytokinins in the female inflorescence. Our data illustrate the importance of branching regulation within the developmental window studied, during which the female inflorescence, unlike its male counterpart, produces flower clusters on new successive axes by sympodial growth.

Environmental regulation of sex determination in oil palm: current knowledge and insights from other species.

BACKGROUND: The African oil palm (Elaeis guineensis) is a monoecious species of the palm subfamily Arecoideae. It may be qualified as 'temporally dioecious' in that it produces functionally unisexual male and female inflorescences in an alternating cycle on the same plant, resulting in an allogamous mode of reproduction. The 'sex ratio' of an oil palm stand is influenced by both genetic and environmental factors. In particular, the enhancement of male inflorescence production in response to water stress has been well documented. SCOPE: This paper presents a review of our current understanding of the sex determination process in oil palm and discusses possible insights that can be gained from other species. Although some informative phenological studies have been carried out, nothing is as yet known about the genetic basis of sex determination in oil palm, nor the mechanisms by which this process is regulated. Nevertheless new genomics-based techniques, when combined with field studies and biochemical and molecular cytological-based approaches, should provide a new understanding of the complex processes governing oil palm sex determination in the foreseeable future. Current hypotheses and strategies for future research are discussed.

Epigenetic imbalance and the floral developmental abnormality of the in vitro-regenerated oil palm Elaeis guineensis.

BACKGROUND: The large-scale clonal propagation of oil palm (Elaeis guineensis) is being stalled by the occurrence of the mantled somaclonal variation. Indeed, this abnormality which presents a homeotic-like conversion of male floral organs into carpelloid structures, hampers oil production since the supernumerary female organs are either sterile or produce fruits with poor oil yields. SCOPE: In the last 15 years, the prevailing point of view on the origin of the mantled floral phenotype has evolved from a random mutation event triggered by in vitro culture to a hormone-dependent dysfunction of gene regulation processes. In this review, we retrace the history of the research on the mantled variation in the light of the parallel advances made in the understanding of plant development regulation in model systems and more specifically in the role of epigenetic mechanisms. An overview of the current state of oil palm genomic and transcriptomic resources, which are key to any comparison with model organisms, is given. We show that, while displaying original characteristics, the mantled phenotype of oil palm is morphologically, and possibly molecularly, related to MADS-box genes mutants described in model plants. We also discuss the occurrence of comparable floral phenotypes in other palm species. CONCLUSIONS: Beyond its primary interest in the search for discriminating markers against an economically crippling phenotype, the study of the mantled abnormality also provides a unique opportunity to investigate the regulation of reproductive development in a perennial tropical palm. On the basis of recent results, we propose that future efforts should concentrate on the epigenetic regulation targeting MADS-box genes and transposable elements of oil palm, since both types of sequences are most likely to be involved in the mantled variant phenotype.

Modulating rice stress tolerance by transcription factors.

Plants are non-mobile organisms and have to adapt to environmental stresses mostly by modulating their growth and development in addition to physiological and biochemical changes. Transcription factors (TFs) regulate genome expression in response to environmental and physiological signals, and some of them switch on plant adaptive developmental and physiological pathways. One TF is encoded by a single gene but regulates the expression of several other genes leading to the activation of complex adaptive mechanisms and hence represents major molecular targets to genetically improve the tolerance of crop plants against different stresses. In this review an updated account of the discovery of TFs involved in biotic and abiotic stress tolerance in the model monocotyledonous plant, rice (Oryza sativa L.) is presented. We illustrate how the elucidation of the function of these TFs can be used to set up genetic engineering strategies and to rationalize molecular breeding using molecular assisted selection towards enhancement of rice tolerance to various stresses. Attempts have also been made to provide information on the molecular mechanisms involved in stress resistance or tolerance processes. We discuss how the comparison of the action of TFs isolated from the dicotyledonous model plant Arabidopsis thaliana in rice and vice versa can contribute to determine whether common or divergent mechanisms underlie stress tolerance in the two plant species. Lastly, we discuss the necessity to discover TFs controlling specifically the root adaptive development which constitutes a major way for the plant to escape to several stresses such as water deficit or mineral nutrient deficiency.

Somaclonal variation in micropropagated oil palm. Characterization of two novel genes with enhanced expression in epigenetically abnormal cell lines and in response to auxin.

In vitro micropropagation based on somatic embryogenesis provides an efficient means to multiply selected genotypes of oil palm (Elaeis guineensis Jacq.). Despite its considerable potential, somatic embryogenesis can yield plants bearing a homeotic flowering abnormality known as mantled. Because the mantled abnormality is epigenetic, it cannot be detected with conventional structural molecular markers. Thus, to develop a means of discriminating among callus cultures carrying or lacking the mantled abnormality, we used a gene expression approach. We describe two novel oil palm genes, EgM39A and EgIAA1, both of which display increased transcript accumulation in epigenetically abnormal calli. EgIAA1 codes for an oil palm relative of the Arabidopsis thaliana (L.) Heynh. AXR3/IAA17 protein involved in early auxin response and EgM39A codes for a protein of unknown function sharing sequence similarities with asparagine synthetases. In addition to their enhanced expression in somaclonal variant callus lines, both genes displayed increased transcript accumulation in response to auxin treatment. Normal seed-derived zygotic embryos germinated in the presence of auxin accumulated increased amounts of EgIAA1 transcripts after a few hours of treatment, suggesting a role in auxin response similar to that demonstrated for IAA genes in other species. The EgM39A gene also displayed enhanced transcript accumulation in auxin-treated zygotic embryos. Although only a small increase was seen after 24 h, greater changes were observed after 15 days. Both genes show potential as early markers of clonal conformity and may help to elucidate the nature of the epigenetic changes causing the mantled abnormality.

DNA methylation and expression of the EgDEF1 gene and neighboring retrotransposons in mantled somaclonal variants of oil palm.

The mantled floral phenotype of oil palm (Elaeis guineensis) affects somatic embryogenesis-derived individuals and is morphologically similar to mutants defective in the B-class MADS-box genes. This somaclonal variation has been previously demonstrated to be associated to a significant deficit in genome-wide DNA methylation. In order to elucidate the possible role of DNA methylation in the transcriptional regulation of EgDEF1, the APETALA3 ortholog of oil palm, we studied this epigenetic mark within the gene in parallel with transcript accumulation in both normal and mantled developing inflorescences. We also examined the methylation and expression of two neighboring retrotransposons that might interfere with EgDEF1 regulation. We show that the EgDEF1 gene is essentially unmethylated and that its methylation pattern does not change with the floral phenotype whereas expression is dramatically different, ruling out a direct implication of DNA methylation in the regulation of this gene. Also, we find that both the gypsy element inserted within an intron of the EgDEF1 gene and the copia element located upstream from the promoter are heavily methylated and show little or no expression. Interestingly, we identify a shorter, alternative transcript produced by EgDEF1 and characterize its accumulation with respect to its full-length counterpart. We demonstrate that, depending on the floral phenotype, the respective proportions of these two transcripts change differently during inflorescence development. We discuss the possible phenotypical consequences of this alternative splicing and the new questions it raises in the search for the molecular mechanisms underlying the mantled phenotype in the oil palm.

Functional characterization of MADS box genes involved in the determination of oil palm flower structure.

In order to study the molecular regulation of flower development in the monoecious species oil palm (Elaeis guineensis), cDNAs of 12 MADS box genes from this plant belonging to seven distinct subfamilies were previously isolated and characterized. Here studies carried out on five of these genes, each likely to be involved in floral morphogenesis: EgSQUA1 (SQUAMOSA subfamily); EgAGL2-1 (AGL2 subfamily); EgGLO2 (GLOBOSA subfamily); EgDEF1 (DEFICIENS subfamily); and EgAG2 (AGAMOUS subfamily), are described. In order to determine where and when in the plant these genes are likely to function, their spatial and temporal patterns of expression were studied during the development of male and female inflorescences, either of normal phenotype or displaying a homeotic flowering abnormality known as mantled. In parallel, the phenotypic effects of ectopically expressing these genes in transgenic Arabidopsis thaliana plants were analysed. The data suggest a broad conservation of floral homeotic gene functions between oil palm and previously described model species, although a few minor variations in the zones of activity of certain genes cannot be excluded. The data also indicate distinct molecular identities for the morphologically similar floral organs of whorls 1 and 2. They also reveal reduced expression of putative B, C/D, and E class genes in mantled flowers, which undergo a homeotic transformation comparable to B class mutants of model species.

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.

Characterization of a defensin gene expressed in oil palm inflorescences: induction during tissue culture and possible association with epigenetic somaclonal variation events.

From differential display studies performed on oil palm (Elaeis guineensis Jacq.) tissue cultures bearing or lacking an epigenetic homeotic flowering abnormality, known as mantled, EGAD1, a gene coding for a putative plant defensin, has been identified and characterized. In whole plants, transcripts of the EGAD1 gene were detected only in inflorescences. The closest characterized relative of the oil palm EGAD1 gene is the Petunia PPT gene, which is expressed principally in the pistil of the flower. The 77 amino acid polypeptide encoded by the EGAD1 gene displays strong similarities with a number of plant defensin proteins, which are thought to play a protective role and which have been shown in some cases to possess antifungal properties. Oil palm tissue cultures exhibit a generally strong induction of accumulation of EGAD1 transcripts, which were detected to differing extents at all stages of the tissue culture regeneration process. The 5' flanking region of the EGAD1 gene was found to contain two different types of potential cis-acting DNA element previously identified in the promoters of plant defence-related genes, which may explain the observed expression in tissue cultures. At the callus stage of the in vitro regeneration procedure, a differential accumulation of EGAD1 transcripts was observed which correlated with the presence or absence of the mantled flowering abnormality. EGAD1 gene expression may therefore be a marker of epigenetic somaclonal variation events.