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  • Open access
  • 68 Reads
Transcriptome Analysis in Cork Oak Using Laser Microdissection and RNA-Seq

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.


  1. Pereira, H. (2007). Cork - Biology, Production and Uses. Elsevier.
  2. Graça, J. (2015). Suberin: The biopolyester at the frontier of plants. Frontiers in Chemistry, 3(OCT), 1–11.
  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.
  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.

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.

  • Open access
  • 23 Reads
Molecular Analysis in Cactus Fruit (Opuntia sp.) Development

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.

  • Open access
  • 34 Reads
Quercus suber transcriptome analyses: identification of genes and SNPs related to cork quality

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.


[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.

  • Open access
  • 55 Reads
MicroRNA-based and proteomic fingerprinting of Avena sativa L. genotypes

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.

  • Open access
  • 60 Reads
Establishment and Optimization of Micrografting Assays with Almond (Prunus dulcis) Portuguese Varieties

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.


[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.

  • Open access
  • 48 Reads
Quantification and Immunolocalization of Auxin in Prunus dulcis (Mill.) D. A. Webb Micrografts

In recent years almond production has increased due to the strong tendency of consumers towards plant-based products. The grafting propagation method is the most widely used for this Prunus species to increase production and fruit quality. During grafting, the plant vasculature is severely damaged thus affecting auxin transport. However, once the scion and the rootstock join, a complex process involving cell division and differentiation of vascular tissues establishes communication between the two parts. Nevertheless, wound healing and auxin-induced regulatory mechanisms involved in scion-rootstock interactions remain largely unknown. Thus, this work aimed to quantify and immunolocalize IAA (indole-3-acetic acid) in almond trees micrografts, before and 21 days after micrografting, an in vitro technique that allows a rapid graft union formation using in vitro established scions and rootstocks.

To achieve this goal, scions and rootstocks were in vitro established and micropropagated. Micrografts were successfully achieved from bitter almond homografts and Canhota (Portuguese traditional variety) x bitter almond rootstocks. IAA quantification performed through Ehrlich reaction, showed a scion content of 1.292±0.448 μg IAA/ mg FW (Canhota) and 5.505±1.179 μg IAA/ mg FW (bitter almond) before micrografting, and the potential influence of these levels on micrograft success. Through IAA immunolocalization a possible accumulation at the graft union in the scion part was observed. The results obtained here are a step forward to the understanding of how scion and rootstock communicate in different almond tree micrograft combinations and how their communication is associated with graft success.

  • Open access
  • 35 Reads
Tocopherol Biosynthesis Dynamics in Almond Kernel Development

Almond is one of the most important tree nut crops, due to its high nutritive value, namely in lipids and tocopherol richness. Its production in Portugal has been increasing during the last years. Tocopherol (Vitamin E) is an important antioxidant that prevents the peroxidation of unsaturated fatty acids, increasing storage life of almonds. It is highly effective in preventing cardiovascular diseases by inhibiting platelet aggregation. Genes involved in its biosynthesis have been characterized in other species, but few studies have been made in almonds.

The aim of this work was the study of change in tocopherol content during almond kernel maturation and molecular mechanisms underlying its biosynthetic pathway.

Different stages of kernel development of variety Soleta were morphologically characterized from March to August in Alentejo region, Portugal. The tocopherol profile was analyzed and quantified by HPLC, using fluorescence detection and a normal-phase silica column. For analysis of transcript abundance by RT-qPCR, total RNA extractions of almond kernels were optimized, followed by synthesized cDNA. Sequence transcripts of vte2, vte3 and vte4, involved in tocopherol synthesis, were confirmed by Sanger sequencing.

It was possible to observe a progressive increase in α-tocopherol content in kernels development over the months, with higher increments in the mature stages. In the future, the differential expression level of candidate genes involved in tocopherol synthesis will be characterized using RT-qPCR. This study intends to reveal new insight into the tocopherol biosynthetic pathway which may impact kernel composition.

  • Open access
  • 67 Reads
Response of Cucumis sativus to Spirotetramat Application Leads to Changes in Salicylic Acid, Antioxidative Enzymes, Amino Acids, Mineral Elements, and Soluble Carbohydrates

Plants are influenced by pesticides in terms of various physio-biochemical parameters. This study provides initial evidence on the effect of the insecticide spirotetramat on plant physiological characteristics as a non-target organism. Cucumber plants (Cucumis sativus L.) exposed to spirotetramat were studied 10 days after treatment. There was an increase in the activity of antioxidant enzymes including superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase, and phenylalanine ammonia-lyase. Malondialdehyde, total chlorophyll, hydrogen peroxide contents, and electrolyte leakage index was not affected by spirotetramat. Further biochemical analyses revealed an increase in the content of some free amino acids, as well as sucrose, glucose, and fructose. The amount of salicylic acid and also minerals calcium, manganese, copper, zinc, iron, nitrogen, and magnesium, were elevated in spirotetramat-treated plants. Results have shown that spirotetramat can manipulate cucumber plant physiology through induces biochemical responses that are reflected in changes of antioxidative enzymes, amino acids, soluble carbohydrates, salicylic acid, and mineral elements. The findings of this study provide an insight into the side effects of spirotetramat as a chemical with no specific target site in plants that show no adverse effects on plant health indices. This study focuses on observed physiological changes related to toxicity in plants exposed to hazardous substances present in the environment that can improve our knowledge and understanding of the underlying effects of xenobiotics on plants.

  • Open access
  • 113 Reads
Aspect and Rootstock Genotype Influence Leaf Stomatal Density in Macadamia sp.

Stomata, the pores on the leaf epidermis, contribute to the exchange of water and CO2 in plants. They play important roles in sensing and adaptation to environmental changes by altering in size, distribution, and behaviour. To adapt to a changing climate, planting cultivars with optimised stomatal density in the target environments can be useful to improve water use efficiency. As a clonally propagated crop, orchard production of macadamia largely depends on rootstocks. The effect of rootstocks on stomatal density in macadamia remains unclear. This study, for the first time in macadamia, aimed to investigate the effects of aspect and rootstock genotype on leaf stomatal density. We investigated the effects of three rootstocks (Beaumont, Daddow and B-29), and two aspects (north (N) and south (S)), on leaf stomatal density. An existing phenotyping protocol was adapted to create an imprint of the leaf surface. This imprint was mounted to a microscope slide for imaging and stomatal density calculation using manual (M) and artificial intelligence (AI) methods. A highly significant interaction was observed between rootstock and stomatal density (P < 0.001 (M), P = 0.003 (AI)). Additionally, stomatal density was found to be significantly higher on the northern aspect of the tree than the southern aspect (P < 0.001 (M), P < 0.001 (AI)). These findings will assist in the development of climate-smart macadamia varieties with superior water use efficiency.

  • Open access
  • 38 Reads
Metabolites Differentiating Asymptomatic and Symptomatic Grapevine Plants (Vitis vinifera ‘Malvasia-Fina’) Infected with Esca Complex Disease-Associated Fungi

Esca complex disease is a grapevine disease mainly caused by the fungi Fomitiporia mediterranea, Phaeomoniella chlamydospora, and Phaeoacremonium minimum. Colonization by esca-associated fungi is restricted to the canes, spurs, cordons, and trunks, and the disease usually exhibits a latency time of 4 to 10 years between wood invasion by fungi and visible foliar symptoms. The goals of this study were to identify metabolites differentiating asymptomatic and symptomatic grapevine leaves and to gain better insights into the mechanisms involved in the delayed appearance of foliar symptoms. Experiments were performed using healthy, asymptomatic and symptomatic leaves of Vitis vinifera L. ‘Malvasia-fina’ naturally infected in the vineyard. A global metabolic profile of the samples was obtained with ultrahigh performance liquid chromatography system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap high resolution/accurate mass spectrometer. In total, 513 metabolites were identified in the leaves, including 436 compounds of known identity and 77 compounds of unknown structural identity, belonging to 9 biochemical families (amino acids, carbohydrates, lipids, cofactors + prosthetic groups + electron carriers, nucleotides, peptides, hormones, secondary metabolites, and xenobiotics). Hormone and lipid data showed that systemic signals are transferred from the infected wood to the leaves. Secondary metabolites data indicated that defence compounds are mostly locally induced following the onset of foliar symptoms. Several primary metabolites showed interesting changed patterns related to the modulation of grapevine metabolism. Random Forest analysis could classify the samples with 96% accuracy, which allowed selecting 30 metabolites with the largest contribution to the differentiation of leaf groups.