Laser Microdissection of Woody and Suberized Plant Tissues for RNA-Seq Analysis.

An accurate profile of gene expression at a cellular level can contribute to a better understanding of biological processes and complexities involved in regulatory mechanism of woody plants. Laser microdissection is one technique that allows isolation of specific, target cells or tissue from a heterogeneous cell population. This technique entails microscopic visualization of the selected tissue and use a laser beam to separate the desired cells from surrounding tissue. Initial identification of these cells is made based on morphology and/or histological staining. Some works have been made in several tissues and plant models. However, there are few studies of laser microdissection application in woody species, particularly, lignified and suberized cells. Moreover, the presence of high level of suberin in cell walls can be a big challenge for the application of this approach. In our study it was developed a technique for tissue isolation, using laser microdissection of four different plant cell types (phellogen, lenticels, cortex and xylem) from woody tissues of cork oak (Quercus suber), followed by RNA extraction and RNA-Seq. We tested several methodologies regarding laser microdissection, cryostat equipments, fixation treatments, duration of single-cells collection and number of isolated cells by laser microdissection and RNA extraction procedures. A simple and efficient protocol for tissue isolation by laser microdissection and RNA purification was obtained, with a final method validation of RNA-Seq analysis. The optimized methodology combining RNA-Seq for expression analysis will contribute to elucidate the molecular pathways associated with different development processes of the xylem and phellem in oaks, including the lenticular channels formation.

Patterns of Volatile Diversity Yield Insights Into the Genetics and Biochemistry of the Date Palm Fruit Volatilome.

Volatile organic compounds are key components of the fruit metabolome that contribute to traits such as aroma and taste. Here we report on the diversity of 90 flavor-related fruit traits in date palms (Phoenix dactylifera L.) including 80 volatile organic compounds, which collectively represent the fruit volatilome, as well as 6 organic acids, and 4 sugars in tree-ripened fruits. We characterize these traits in 148 date palms representing 135 varieties using headspace solid-phase microextraction gas chromatography. We discovered new volatile compounds unknown in date palm including 2-methoxy-4-vinylphenol, an attractant of the red palm weevil (Rhynchophorus ferrugineus Olivier), a key pest that threatens the date palm crop. Associations between volatile composition and sugar and moisture content suggest that differences among fruits in these traits may be characterized by system-wide differences in fruit metabolism. Correlations between volatiles indicate medium chain and long chain fatty acid ester volatiles are regulated independently, possibly reflecting differences in the biochemistry of fatty acid precursors. Finally, we took advantage of date palm clones in our analysis to estimate broad-sense heritabilities of volatiles and demonstrate that at least some of volatile diversity has a genetic basis.

ChIP-Seq reveals that QsMYB1 directly targets genes involved in lignin and suberin biosynthesis pathways in cork oak (Quercus suber).

BACKGROUND: Gene activity is largely controlled by transcriptional regulation through the action of transcription factors and other regulators. QsMYB1 is a member of the R2R3-MYB transcription factor family related to secondary growth, and in particular, with the cork development process. In order to identify the putative gene targets of QsMYB1 across the cork oak genome we developed a ChIP-Seq strategy. RESULTS: Results provide direct evidence that QsMY1B targets genes encoding for enzymes involved in the lignin and suberin pathways as well as gene encoding for ABCG transporters and LTPs implicated in the transport of monomeric suberin units across the cellular membrane. These results highlight the role of QsMYB1 as a regulator of lignin and suberin biosynthesis, transport and assembly. CONCLUSION: To our knowledge, this work constitutes the first ChIP-Seq experiment performed in cork oak, a non-model plant species with a long-life cycle, and these results will contribute to deepen the knowledge about the molecular mechanisms of cork formation and differentiation.

The draft genome sequence of cork oak.

Cork oak (Quercus suber) is native to southwest Europe and northwest Africa where it plays a crucial environmental and economical role. To tackle the cork oak production and industrial challenges, advanced research is imperative but dependent on the availability of a sequenced genome. To address this, we produced the first draft version of the cork oak genome. We followed a de novo assembly strategy based on high-throughput sequence data, which generated a draft genome comprising 23,347 scaffolds and 953.3 Mb in size. A total of 79,752 genes and 83,814 transcripts were predicted, including 33,658 high-confidence genes. An InterPro signature assignment was detected for 69,218 transcripts, which represented 82.6% of the total. Validation studies demonstrated the genome assembly and annotation completeness and highlighted the usefulness of the draft genome for read mapping of high-throughput sequence data generated using different protocols. All data generated is available through the public databases where it was deposited, being therefore ready to use by the academic and industry communities working on cork oak and/or related species.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Molecular characterization of Quercus suber MYB1, a transcription factor up-regulated in cork tissues.

The molecular processes associated with cork development in Quercus suber L. are poorly understood. A previous molecular approach identified a list of genes potentially important for cork formation and differentiation, providing a new basis for further molecular studies. This report is the first molecular characterization of one of these candidate genes, QsMYB1, coding for an R2R3-MYB transcription factor. The R2R3-MYB gene sub-family has been described as being involved in the phenylpropanoid and lignin pathways, both involved in cork biosynthesis. The results showed that the expression of QsMYB1 is putatively mediated by an alternative splicing (AS) mechanism that originates two different transcripts (QsMYB1.1 and QsMYB1.2), differing only in the 5'-untranslated region, due to retention of the first intron in one of the variants. Moreover, within the retained intron, a simple sequence repeat (SSR) was identified. The upstream regulatory region of QsMYB1 was extended by a genome walking approach, which allowed the identification of the putative gene promoter region. The relative expression pattern of QsMYB1 transcripts determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed that both transcripts were up-regulated in cork tissues; the detected expression was several times higher in newly formed cork harvested from trees producing virgin, second or reproduction cork when compared with wood. Moreover, the expression analysis of QsMYB1 in several Q. suber organs showed very low expression in young branches and roots, whereas in leaves, immature acorns or male flowers, no expression was detected. These preliminary results suggest that QsMYB1 may be related to secondary growth and, in particular, with the cork biosynthesis process with a possible alternative splicing mechanism associated with its regulatory function.