Cork from Quercus suber L. is a non-wood forest product with high economic and commercial value, as well social and ecological role. In Portugal, cork represents 33% of all national forestry products placing Portugal as the world leader in cork production, industrial processing, and trade of cork. The impermeability, fire retardancy, and sound insulation properties of cork make it the optimum material for a variety of applications, such as wine bottles stoppers, insulation corkboard, shoe soles, and others fashion purposes [1, 2].

Laser microdissection microscopy (LM) combined with RNA-sequencing are powerful techniques for investigating the transcriptome profile of specific tissues or cell types at a cellular level [3].

In order to identify specific candidate genes linked to secondary growth, a transcriptomic analysis of single-cells isolation of cork oak, by LM technology was performed. Thus, an optimized protocol for single-cell isolation by LM in suber-phellogen and xylem was successfully obtained, followed by RNA isolation and cDNA libraries preparation and RNA sequencing. High-quality reads (MAPQ>20) and a minimum size of 130 bp were obtained, followed by alignment and mapping against the cork oak genome [4]. About 25 to 30 million reads were uniquely mapped to the cork oak genome sequence. The mapping results suggest that single-cell isolation, RNA extraction, and sequencing of Illumina libraries procedures were viable for transcriptomic studies of cork oak tissues. The scRNA-Seq will allow gene expression analysis in individual tissues of oaks, contributing to understand the molecular mechanisms associated with the development processes of secondary growth.

References:

1. Pereira, H. (2007). Cork - Biology, Production and Uses. Elsevier. https://doi.org/https://doi.org/10.1016/B978-0-444-52967-1.X5000-6
2. Graça, J. (2015). Suberin: The biopolyester at the frontier of plants. Frontiers in Chemistry, 3(OCT), 1–11. https://doi.org/10.3389/fchem.2015.00062
3. Gautam, V., & Sarkar, A. K. (2015). Laser Assisted Microdissection, an Efficient Technique to Understand Tissue Specific Gene Expression Patterns and Functional Genomics in Plants. Molecular Biotechnology, 57(4), 299–308. https://doi.org/10.1007/s12033-014-9824-3
4. Ramos, A. M., Usié, A., Barbosa, P., Barros, P. M., Capote, T., Chaves, I., … Gonçalves, S. (2018). The draft genome sequence of cork oak. Scientific Data, 5, 1–12. https://doi.org/10.1038/sdata.2018.69

Acknowledgments: This work was supported by Alentejo2020, through FEDER under Lentidev- “A molecular approach to cork porosity” project (ALT20-03-0145-FEDER-000020). Authors also acknowledge FCT for Contrato – Programa to L. Marum (CEECINST/00131/2018) and FCT for UIDB/05183/2020.

Nopal is an originary plant from Mexico, it can growth under extreme conditions, it is also an ancestral crop that help to establish the mexican civilization. Nopal and its products, including fruits are considered a key crop for food sufficiency and security in Mexico. Prickly pear (cactus fruit) shows great variability in its morphology, however there are two disadvantages: the number and size of seeds and thorns; it will be interesting to identify the molecular processes associated to these characteristics. the main objective of this work is to identify the molecular mechanisms that regulate the development of prickly pear. Tissues from four stages were collected; flowers, fertilized button, green and ripe fruit from an intermediate ripening morphospecie (Reina). miRNA expresión at this stages was analyzed with a microarray and a transcriptome expression, computational analysis helped to identify and select unique miRNAs from each developmental stage. A total of 43 miRNAs with 26 different targets were identified in relation to fruit development from flower induction. It highlights the importance of miR172 and miR395, as key candidates in the fruit ripening time. miR397 is also established, as an opportunity for the generation of varieties for control of seed production. The mechanism of action of microRNAs was tested in other plants successfully.

Cork is an ancestral natural material derived from the cork oak tree, with multiple industrial applications [1]. The cork quality, one of the main concerns of cork industry, strongly influences the market value of this non-wood product, which plays an important role in the Mediterranean economies. It is known that cork quality is dependent on both genetic and environmental factors. The recent sequencing of the cork oak genome allowed a very comprehensive characterization of the species’ genome and opened the possibility to execute numerous studies to appraise old and new questions that cork production continues to raise [2].

In this study, the transcriptome of phellogen tissue harvested from cork oak trees producing good and bad quality cork was performed using Illumina sequencing data in order to elucidate differentially expressed genes (DEGs), gene ontology (GO), and functional pathway analysis. Additionally, a variant calling analyses was performed in order to identify differences between trees from each group.

The results revealed the presence of highly-expressed genes involved in stress response, transmembrane transport and proteolysis, in trees producing cork of bad quality. On the other hand, genes involved in the fatty acid biosynthetic pathway, resulting in important precursors of waxes and some suberin monomers, were highly expressed in trees producing good quality cork. A total of 296,640 variants were identified of which 159,248 were high-quality Single Nucleotide Polymorphisms (SNPs).

This study highlights a set of mechanisms that the trees activate during cork differentiation, and which can influence the cork quality.

References:

[1] Duarte AP and Bordado JC (2015) Cork – a renewable raw material: forecast of industrial potential and development priorities. doi: 10.3389/fmats.2015.00002

[2] Ramos AM, Usié A, Barbosa P, Barros PM, Capote T, Chaves I, Simões F, Abreu I, Carrasquinho I, Faro C, et al (2018) The draft genome sequence of cork oak. Sci data 5: 180069

Acknowledgements: Work was supported by InAlentejo under the scope “GenoSuber - Cork oak genome sequencing” (ALENT-07-0224-FEDER-001754) and by Alentejo2020, through FEDER under the scope “Lentidev - A genomic approach to cork quality” (ALT20-03-0145-FEDER-000020). Authors also acknowledge FCT for Contrato – Programa to L. Marum (CEECINST/00131/2018) and FCT for UIDB/05183/2020.

MicroRNA (miRNA) molecules have a broad-spectrum effect due to their regulatory potential in almost all biological and metabolic processes including plant stress adaptation mechanism. Due to their high conservation, sequences of miRNA provide an effective type of putative functional markers which responsiveness may be species-, tissues/organs- and genotype-specific. Genomic characterization of 15 common oat genotypes of various origin was performed by several miRNA-based markers (miR156, miR398, miR399, miR408, miR827 and miR858). The marker miR408 showed the highest polymorphism and the highest amplification rate (almost 27% of all amplified loci). This type of miRNAs is part of the regulatory mechanisms of photosynthesis and processes related to biomass production. Markers related to nitrogen (miR827), phosphorus (miR399) and copper (miR398) metabolism together account for 47% of amplified miRNA loci. Markers related to flavonoid biosynthesis (miR156 and miR858) together account for 26%. In Sweden genotypes (Arne and Magne) was recorded the highest amplification of markers loci in general. MiRNA-based markers enabled screening of Avena sativa genomic polymorphism. By proteomics approaches (SDS and A-PAGE electrophoresis) was detected sufficient diversity between the analyzed samples, and genetic related dendrograms were constructed based on the electrophoretic profiles.

Almond is a nutritive fruit, rich in monounsaturated fats, protein, and antioxidants. In Portugal, almonds are a traditional culture in the north and south. However, new orchards are being installed mainly in irrigated systems using commercial varieties neglecting the traditional cultivars. Micrografting, grafting in vitro conditions, involves the placement of a shoot explant into a decapitated rootstock in sterilized conditions ^[1]. This technique allows rapid and large-scale multiplication, and it has been used for the production of disease-free plants, rejuvenation of mature tissues, and to determine the compatibility between the scion and the rootstock. Only a few protocols have been established for commercial almond trees ^[2], but no studies have been described with Portuguese varieties.

The main objective of this work was to optimize new and efficient protocols of almond micrografting with traditional varieties (Rabo de Zorra, Gama Dura and Canhota), known for their high fruit quality. The effect of plant growth regulators (BAP and IBA) and activated charcoal on culture medium were evaluated during micrografting assays. In addition, rooting assays of bitter almond rootstock were also analysed. The healing percentage of micrografts observed was higher than 87%. Auxin IBA was important for root induction of rootstocks during micrografting and rootstock assays. However, the IBA dipping procedure (IBA 1 mg/L) presented higher rooting rates (60%), when compared to MS medium supplement with IBA (27%) in the same concentrations. In the future, the success of almond micrografts will be evaluated in ex-vitro conditions, namely in field tests.

Reference:

[1] Hussain, G., Wani, M.S., Mir, M.A., Rather, Z.A., Bhat, K. M., (2014). Micrografting for fruit crop improvement. African Journal of Biotechnology, 13, 2474-2483.

[2] Yildirim, H., Akdemir, H., Süzerer, V., Ozden, Y., Onay, A., (2013). In Vitro Micrografting of the almond Cultivars “Texas”, “Ferrastar” and “Nonpareil”. Biotechnology & Biotechnological Equipment, 27:1, 3493-3501.