Applications of Systems Biology

**65**

**Chapter 5**

**Abstract**

PCR screening

**1. Introduction**

Application of Genomic Data

for PCR Screening of Bet v 1

Relevant Plant Species

*Jana Žiarovská and Lucia Zeleňáková*

45 clinically relevant plant species.

Conserved Sequence in Clinically

Bet v 1 is a highly immunogenic protein, which is the main cause of sensitivity to birch pollen and is described as the main birch allergen. Despite the structural similarity, Bet v 1 homologs show different properties and immunoreactivity. Here, the bioinformatic algorithms were applied for known Bet v 1 homologous nucleic acids sequences to find homology and conserved regions. Genomic sequences of PR proteins of two different fruit species, which allergens belong to PR proteins of the same type as Bet v 1, were selected to design degenerate primers. Subsequently, screening of the presence of Bet v 1 conserved genomic sequence was performed in

**Keywords:** genomic sequences, Bet v 1, conserved region, degenerate primers,

related protein family (PR-10) is expressionally connected to the pathogen attack or abiotic stress. Highest concentrations of PR-10 proteins were found in reproductive tissues (pollen, seeds and fruits) [3] and were described with a high level of similarity with the human lipocalin 2. Birch Bet v 1 and human lipocalin 2 possess specific structures that allowed them to bind iron. Bet v 1 turns to in the situation when it is not binding iron. This subsequently affects Th2 cells of the human immune system [4]. The ribonuclease activity of PR proteins is known to be activated under the function in antiviral pathway [5]. The other subfamilies of

Genomic knowledge about major birch pollen allergen is very well known for quite a long time. In the last 30 years, many of different homologs for Bet v 1 have been cloned, and many of their products were characterized from the allergenic point of view. Molecular profiling of allergic sensitization has helped to elucidate the immunological connections of allergen cross-reactivity, whereas advances in biochemistry have revealed structural and functional aspects of allergenic proteins in the last decades [1]. Bet v 1 has been identified as existing in three subfamilies, based on the sequence similarity. The most precise identification is actually done for major birch allergen Bet v 1 that was firstly identified in *Betulla verrucosa* [2]. Bet v 1 is reported in vascular plants as common ones. The first class, pathogenesis-

#### **Chapter 5**

## Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically Relevant Plant Species

*Jana Žiarovská and Lucia Zeleňáková* 

### **Abstract**

Bet v 1 is a highly immunogenic protein, which is the main cause of sensitivity to birch pollen and is described as the main birch allergen. Despite the structural similarity, Bet v 1 homologs show different properties and immunoreactivity. Here, the bioinformatic algorithms were applied for known Bet v 1 homologous nucleic acids sequences to find homology and conserved regions. Genomic sequences of PR proteins of two different fruit species, which allergens belong to PR proteins of the same type as Bet v 1, were selected to design degenerate primers. Subsequently, screening of the presence of Bet v 1 conserved genomic sequence was performed in 45 clinically relevant plant species.

**Keywords:** genomic sequences, Bet v 1, conserved region, degenerate primers, PCR screening

#### **1. Introduction**

 Genomic knowledge about major birch pollen allergen is very well known for quite a long time. In the last 30 years, many of different homologs for Bet v 1 have been cloned, and many of their products were characterized from the allergenic point of view. Molecular profiling of allergic sensitization has helped to elucidate the immunological connections of allergen cross-reactivity, whereas advances in biochemistry have revealed structural and functional aspects of allergenic proteins in the last decades [1]. Bet v 1 has been identified as existing in three subfamilies, based on the sequence similarity. The most precise identification is actually done for major birch allergen Bet v 1 that was firstly identified in *Betulla verrucosa* [2]. Bet v 1 is reported in vascular plants as common ones. The first class, pathogenesisrelated protein family (PR-10) is expressionally connected to the pathogen attack or abiotic stress. Highest concentrations of PR-10 proteins were found in reproductive tissues (pollen, seeds and fruits) [3] and were described with a high level of similarity with the human lipocalin 2. Birch Bet v 1 and human lipocalin 2 possess specific structures that allowed them to bind iron. Bet v 1 turns to in the situation when it is not binding iron. This subsequently affects Th2 cells of the human immune system [4]. The ribonuclease activity of PR proteins is known to be activated under the function in antiviral pathway [5]. The other subfamilies of

Bet v 1 allergens are reported as major latex proteins and ripening-related proteins in the latex of opium poppy [6, 7]. The last one is reported to be proteins containing members with S-norcoclaurine synthase activity and is involved in alkaloid biosynthesis [8].

 Bet v 1 belongs to panallergens, specifically to PR-10 proteins. Location of Bet v 1 for IgE recognition is the result of the protein chain composition, coming in close proximity of molecules that are spaced apart from the stretched chain. This conformation is disrupted by heat that is why Bet v 1 is defined as thermolabile and it became nonallergenic by cooking or heat processing of fruit. Different denaturation temperatures of the Bet v 1 allergen exist for different individual isolated homologs and their isoforms. A pH value and other thermodynamic and physicochemical properties have a denaturing effect beside the temperature alone [9]. In general, all the PR-10 proteins are labile proteins when comparing them to most of other food allergens [10]. The naturally occurring Bet in 1 consists of several isoforms with a molecular weight of about 17.5 kDa. These isoforms share a high percentage of the same sequences but may have a very different allergenic potential [11, 12]. There are currently more than 20 isoforms found on the IUIS Allergen Nomenclature subcommittee website (http://www.allergen.org). When regarding a total amount of Bet v 1 in grain pollen, about 35% represents the hyperallergenic isoform of Bet in 1.0101 and this is also described in the literature as Bet v 1a [12, 13]. This isoform plays an important role in the development of allergies. It is characterized as the most allergenic isoform. It is used to produce recombinant proteins [11, 12]. Hypoallergenic isoforms are potential candidates for allergen-specific immunotherapy [14]. Despite the structural similarity, Bet v 1 homologs show different properties and immunoreactivity. An example of this is Bet v 1 l - a hypoallergenic isoform, differing in only nine amino acids from highly allergenic Bet v 1a [15]. It is assumed that these different immunological properties are the result of the changing dynamics and stability of the composition of the molecules [16, 17]. Bet v 1 homology is a typical example of a pollen-food allergy syndrome (**Figure 1**). In this case, homology is very close, epitope matching can be up to 90% [18]. Food hypersensitivity to apple, carrot, hazelnuts or celery has been developed in 50–90% of individuals with pollen allergy due to this cross-reactivity in the moderate environment conditions of Europe [19].

**Figure 1.**  *Clinically relevant Bet v 1 homology.* 

*Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically… DOI: http://dx.doi.org/10.5772/intechopen.80312* 

 Bet v 1 homology is referred to as birch-fruit-vegetables-nuts syndrome. The most common are plants from the family *Rosaceae* and *Apiaceae*. Similarities in amino acid sequences were found in different plants and foods [20, 21] but a fruit similarity prevails (**Table 1**). Most often, allergens are located in fruit pulp. With respect to homology to the main birch allergen Bet v 1, it can be noted that in areas where the incidence of birch is not quite typical, for example, in southern Europe, sensitivity to Bet in 1 homologs occurs in trees that are similar to alder, hazel, beech and grass allergens [22]. Pomegranate, edible chestnuts, raspberries, spices may also be mentioned. Hrubiško et al. [23] mentioned the crossreactivity of birch pollen with walnut, almonds, avocados, cherry, plum, peas or asparagus.

Silver Birch is native in most of Europe, northwest Africa and western Siberia, but absent in the southern parts of Europe. It is the most common tree found in Scandinavia and the Alps and a potent pollen producer in those areas. In all of those areas, birch is the most relevant spring pollen allergen relevant during the period from March to May (**Figure 2**).

Atmospheric concentrations of birch pollen grains were monitored [24] and the matched major birch pollen allergen Bet v 1 simultaneously across Europe. The major birch pollen allergen Bet v 1 was determined with an allergen-specific ELISA. The average European allergen release from birch pollen was 3.2 pg. Bet v 1/ pollen and the average allergen release in 2009 did not differ substantially between countries. However, a>10-fold difference between daily allergen releases per pollen was detected in all countries. Results of aeropalynological observations in Kiev were reported [25] to be carried out with a gravimetric method. The most abundant pollen types were as follows: *Betulaceae* (21%), *Chenopodiaceae* (10%), *Ambrosia*  (10%), *Artemisia* (9%) *Pinaceae* (8%) and *Poaceae* (6%).

A real-time PCR method based on SYBR GREEN technology was developed to analyze the different Bet v 1 expression level [26]. The expression of Betv1 allergen gene was analyzed upon various growth places around Kiev of tested birch pollen samples. Sample from forest growth condition was chosen as a calibrator for


#### **Table 1.**

*Protein homology of Bet v 1.* 

**Figure 2.** 

*Pollen load map of Europe during the April (https://www.polleninfo.org). Pollen counts are differed by color: green—low; yellow—mild; orange—high; red—very high.* 

expression analyses. qRT-PCR showed a variation in the abundance of allergen transcripts among the samples from different places of growth (**Figure 3**). In samples from urbanized area was the expression of Betv1 allergen in average 1.5× higher (ranged from 0.77 up to the 2× higher) when comparing to the forest sample served as a calibrator. In samples from borders of the urbanized area was the expression of Betv1 allergen only 0.55× higher when comparing to the forest sample.

These findings are interesting when comparing them with those findings [24] that reported that extracts from pollen collected in urban areas had higher chemotactic activity on human neutrophils compared to pollen from rural sites, although the allergen content remained unchanged. Questions about the exact correlations between the expression level and allergenic potential need are to be answered in further research.

Actually, different primary genomic data are available for Bet v 1 isoforms originated from birch (**Table 2**) and only limited information exist about its transcriptomic characteristics.

 Beside the Bet v 1 – basic allergen component of birch pollen pelvis, minor components exist as well and some of them are clinically relevant too. Allergens of molecular weights of 29.5, 17, 12.5 and 13 kDa had been isolated form birch pollen. The following allergens have been characterized (except of Bet v 1): Bet v 2, a 15 kDa, a profiling; Bet v 3, a 24 kDa calcium-binding protein; Bet v 4, a 9 kDa calcium-binding protein; Bet v 5, a 35 kDa isoflavone reductase-related protein; Bet v 6, a 30–35 kDa protein, phenylcoumaran benzylic ether reductase; Bet v 7, a 18 kDa protein, a cyclophilin and Bet v 11 (www.phadia.com).

#### **Figure 3.**

*Expression ratios of Bet v 1 for analyzed birch pollen samples from Ukraine [26].* 

*Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically… DOI: http://dx.doi.org/10.5772/intechopen.80312* 


**Table 2.** 

*Available genomic data of birch Bet v 1 isoform stored in public databases.* 

#### **2. Bet v 1 genomics and** *in silico* **analysis**

 From a theoretical point of view, in nature, a protein similarity or analogy to the protein antigen exists to any not only in the plant but also in the animal kingdom. Evidence for this is antigens, particularly those with allergenic potential. From a practical point of view, there is such a similarity for almost every protein and is called homology. Proteins that are similar are referred as the protein family/ superfamily. There are a huge number of protein families, many of which have been confirmed to be with allergenic activity [18]. In homology, it is the result of a common evolutionary origin. Homologous genes can be characterized as two or more genes derived from a common original DNA sequence [27]. When identifying genes in the model species and related species, it is often important to distinguish genes mutually linked directly by the species and genes that have been duplicated independently from them. These are two types of homologous genes, orthologs and paralogs with many definitions of them. A status where homology is the result of gene duplication, that is, the two copies remain side by side during the body's past (e.g., alpha and beta hemoglobin), that is why, the genes are called paralogs (para = parallel, analogous, concurrent). In a situation where homology is the result of speciation, that is, the process of generating species, and the past of the gene reflects the past of the species (e.g., alpha hemoglobin in both humans and mice), it should be about orthologs (ortho = exact). Orthologs are genes that are associated with a common origin, genes of different species that have evolved from a common ancestral gene, are called "true" homologs. These genes tend to maintain the same function as the gene they developed from during development process. The identification of orthologs is crucial for a reliable prognosis of gene function in novel genes. Paralogs are genes associated with duplication in the genome. They develop new features even when they are associated with the original function. They deviate from each other within the species. Unlike orthologs, the paralogs gene is a new gene that has a new function. During gene duplication, one copy of the gene is

**Figure 4.**  *Sequence phylogenesis of Bet v 1 isoallergens.* 

mutated to produce a new gene with a new function, although the function often relates to the role of the generic gene [27–29]. Paralogs may result from different types of gene duplication, unequal crossing-over, transposon-mediated duplication or polyploidy, that is, increase in the number of chromosomes in the cell nucleus above the normal diploid state [29]. In the case of the molecular systematics of organisms, it is desirable that the studied sequences are homologously specific they are called orthologous [30].

Orthologs exist in genomes in a single copy that performs the same function in all organisms examined. The series of evolutionarily conserved genes are paralogs *Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically… DOI: http://dx.doi.org/10.5772/intechopen.80312* 

 during the evolution; they were done with one or more duplications, followed by the separation of the structure and the functions to the loss of some copies. In some organisms (e.g., in higher plants), the determination of orthologs and paralogs is problematic, their genomes have undergone a series of gene duplications and loss of individual copies of genes. Gene duplication is understood as the source of new genes with new features, but it is not always a fundamental transformation of gene function. Duplicated genes often retain a certain degree of functional overlap that is in certain conditions can be manifested as redundancy.

Different Bet v 1 isoforms are relevant as to be naturally existed—a, b, c, d, e, f, g, j and l. When describing the process of the induction of type I allergy, they differ in reaction mechanism with the IgE from patients, and it is reported in [7] that comparison of *in vitro* and *in vivo* IgE binding activity is influenced by the six amino acid residues at different positions of the Bet v 1 molecule. *Betula verrucosa* (*pendula*) Bet v 1 is well known on the nucleotide level, too and 47 isoallergens sequences are stored in the NCBI database for its mRNA with the different level of their sequence identities. Dendrogram of phylogenetic similarity of the Bet v 1 isoallergens sequences with the gene coding Betv1 (X15877.1) is illustrated in **Figure 4**.

#### **3. Technical approaches and methodologies for PCR screening of Bet v 1 sequences in plants**

Bioinformatics provides an interdisciplinary tool, that is used to manage and analyze biological data and known sequences of nucleic acids. Many features of nucleic acids can be used in bioinformatic algorithms as motifs for description of their genomic variability and their better understanding. Individual sequence motifs are recognized by their order and nucleotide preference, and many motif discovery algorithms have been used in different molecular or bioinformatic studies [31–34].

 Here, the bioinformatic algorithms were applied for known Bet v 1 homologous sequences what makes them suitable for applying bioinformatic tools such as BLAST [35] to find homology or conserved regions. The first step was to align the individual isoforms and their variants with each other. First, isoforms that exist in two or three variants in the database were compared to each other, namely Bet v 1.0101, Bet v 1.0102, Bet at 1.0104 and Bet at 1.0204. **Table 3** shows results of the sequence alignment of the variants of the Bet isoform at 1.0101. All three isoforms are linear mRNAs, differing only in the number of base pairs. Records Z80099.1 and Z80098.1 have the same number of base pairs. Their overlaps and query cover are up to 100% and the identity 99%.

 Bet v 1.0102 can be found in the NCBI under the names of *B. verrucosa* Bet v 1d mRNA (mRNA linear and 677 bp) and *B. verrucosa* Bet in 1 h mRNA (also linear mRNA with 677 bp). They possess a 100% query cover and 99% identity using when using the megablast algorithm. Similar, 100% query cover and 99% identity exist for of the Bet v 1.0104 (*B. verrucosa* Bet v 1f mRNA and *B. verrucosa* Bet in 1i mRNA, both with 572 base pairs). Both searches for Bet v 1.0204 in NCBI are mRNA linear, *B. verrucosa* mRNA for the Bet v 1 m isoform has 687 bp, unlike *B. verrucosa* mRNA for Bet v1n, isoform of birch pollen allergen with 737 bp. Their overlap is 91% with 99% match. After a previous comparison, the consistency of the individual sequences can be as very high, so the variants of the isoforms with the highest number of base pairs were used in the next part of the biological analysis.

Using the BLAST algorithm, individual isoforms corresponding to genomic DNA or mRNA sequences were aligned to each other. The following isoforms are DNA sequences: Bet v 1.0115, Bet v 1.0116, Bet at 1.0119, Bet at 1.0205, Bet v 1.0206, Bet at 1.0207. These accessions have a different number of base pairs. An exception from the point of view of the source exist - Bet v 1.0207 (EU526193.1), with the


**Table 3.** 

*Alignment of Bet v 1.0101 nucleotide isoforms that are stored in public databases.* 

source organism *Betula lenta*, bust stiff. The rest of the aligned sequences have the source organism *Betula pendula* (syn. *B. verrucosa*). As isoforms of one allergen, they are very similar to each other (**Table 4**).

Number of nucleotide differences among Bet v 1 isoforms for the conserved part based on the NCBI data are summarized in **Figure 5**.

 The aim for the design of degenerate primers and their subsequent application in the analysis is the basic description and molecular classifications of allergens; finding of correlations between sequence and structural similarities and cross-reactivity between homologous allergens. Genomic knowledge of allergens also helps to define their common properties and will be helping to clear possible factors that cause allergenic potential in the future [37]. Basis necessity in degenerate primer designing is an alignment of selected nucleotide sequences [38]. Here, Bet v 1 was used as a model to analyze functionality of degenerate primers in clinically relevant cross species screening of genomic sequences of allergens (**Table 5**).

Bet v 1 is standardly used as a model pollen protein PR-10 allergen in different types of research aims [12]. Genomic sequences of PR proteins of two different fruit species which allergens belong to PR proteins of the same type as Bet v 1 were selected to design


#### **Table 4.**

*Alignment of described Bet v 1 isoforms.* 


*Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically… DOI: http://dx.doi.org/10.5772/intechopen.80312* 


#### **Table 5.**

*Clinically high important cross-allergy species to birch pollen and its genomic information availability about PR-10 class allergens.* 

#### **Figure 5.**

*Number of nucleotide differences among Bet v 1 isoforms for the conserved part based on the NCBI data. \*No sequence homology found; all the isoforms are compared to: X15877.1 and the sequences are coded as follows: 1—AF124839.1; 2—AF124838.1; 3—AF124837.1; 4—AJ002110.1; 5—AJ002109.1; 6—AJ002108.1; 7—AJ002107.1; 8—AJ002106.1; 9—X77200.1; 10—X77272.1; 11—X77274.1; 12—X77273.1; 13—X77271.1; 14—X77270.1; 15—X77268.1; 16—X77267.1; 17—X77266.1; 18—X77265.1; 19—X77269.1; 20—X77599.1; 21—X77600.1; 22— X77601.1; 23—Y12560.1; 24—AJ006915.1; 25—AJ006914.1; 26—AJ006913.1; 27—AJ006912.1; 28—AJ006911.1; 29—AJ006910.1; 30—AJ006909.1; 31—AJ006908.1; 32—AJ006907.1; 33—AJ006905.1; 34—AJ006904.1; 35—AJ006903.1; 36—AJ006906.1; 37—Z80106.1; 38—Z80105.1; 39—Z80103.1; 40—Z80102.1; 41—Z80101.1; 42— Z80100.1; 43—Z80099.1; 44—Z80098.1; 45—Z80104.1;46—X82028.1; 47—X81972.1 [36].* 

degenerate primers and to find conserved sequence, that is, the base sequence of the DNA molecule that remained essentially unchanged and thus maintained during the development [39]. *Malus domestica* was used in the selection as a typical fruit caused cross-allergy and *Vitis vinifera* was used in the selection as a species with an allergenic protein homology but without a high clinical relevance. Based on the alignment analysis reported above, Bet v 1 promoter exons (**Table 6**) were used in the *in silico* analysis.

*Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically… DOI: http://dx.doi.org/10.5772/intechopen.80312* 


#### **Table 6.**

*Genomic sequences used for the conserved region identification.* 

 These sequences were aligned by BLAST as conservative sequences can be identified by homologous searching using this too [39]. Specifically, blastn was used for inter-species comparisons with the result showed in **Figure 6** where alignment of sequences AJ289771.1, AJ291705.2, AF020542.1 to sequence AJ289770.1 can be seen.

The design of degenerate primers for optimal PCR amplification should be based on a conserved region with a length of approximately 200–500 base pairs [38] what is the length that was positively identified in the screened Bet v 1 homologs. Degenerate primer is a mixture of oligonucleotide sequences, each of which has a slightly different sequence, that is, there are several probable bases in it. This extends the range of sequences that can be amplified. This is a sequence of approximately 20–25 bp in length, but the forward and reverse primers must be sufficiently distant from each other, it is another characteristic that was identified positively in the aligned sequences. Based on the obtained results, degenerate primers were designed in this region (**Figure 7**) that provide an amplicon with the length of approximately 365 bp.

 Plant material of clinically relevant Bet v 1 (high rate and low rate cross-reactions) cross-reactive plant species and spices were used for the PCR screening analysis. Birch DNA was used as a positive control in the analysis. Total genomic DNA was extracted following the instructions of GeneJET Plant Genomic DNA Purification Mini Kit (Thermo Scientific) or NucleoSpin® Food (Macherey-Nagel). Nanodrop Nanophotometer™ was used for quantity and quality analysis of the extracted DNA. PCR amplifications were performed in a Bio-Rad C1000™ Thermocycler with the following program: an initial denaturation step at 95°C for 5 min followed by

#### **Figure 6.**

*Most conserved regions of Bet v 1 homologs in apple and grape.* 

#### **Figure 7.**

*Selected parts from conservative regions in apple and grape homologs of Bet v 1 that were used for forward (A) and reverse (B) primers designation.* 

40 cycles at 95°C for 45 s, 54°C for 45 s, and 72°C for 35 s with a final cycle at 72°C for 10 min. The amplified products were inspected by electrophoresis in 1.5% agarose in a 1 × TBE buffer, visualized after GelRed™ staining and photographed under UV light.

 Using the degenerate primer pair that was designed on the basis of identified conservative region of Bet v 1 sequences, in all of the screened plant species, PCR was positive with the exception of two samples—curry and black pepper spice (**Table 7**). Here, in the case of curry, only a very weak amplicon is visible in the agarose gel (**Figure 8**), that is why it can be supposed, that a further optimization of degenerate PCR will give a positive result, too. In the case of black pepper spice, using an alternative DNA extraction protocols should be tested further.

Sequence homology search algorithms became commonly used and efficient tools in molecular genetics [39, 40]. Nowadays, a number of different motifs finding algorithms are available and reported them to be impossible to provide a comprehensive report of all of them. Each algorithm has its own advantages and disadvantages. One of the aims of different patterns discovery is finding of specific motifs in nucleotide or protein sequences for the purpose of better understanding of their structure and function [41]


**Table 7.** 

*Results of PCR screening of conserved region of Bet v 1 genomic sequence in clinically relevant plant species.* 

*Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically… DOI: http://dx.doi.org/10.5772/intechopen.80312* 

**Figure 8.** 

*Electrophoretogram profiles of amplicons of conserved region of Bet v 1 genomic sequence in clinically relevant plant species.* 

 or for their identification [42]. Describing the existing polymorphism is relevant for allergens not only toward its static description, but moreover toward its biological and clinical implication. Different changes in allergen expression are reported for pollution or abiotic stress responses [26, 43, 44]. Very specific knowledge is obtained in the field of the variability of allergenic molecules with respect to the genetic origin of the allergens for different plant species, such as olive, palm date or apple [45–48]. In birch, 13 Bet v 1 putative alleles have been characterized and their occurrence in different cultivars is a matter for future study [49]. Allergens identification has become an integral part of the characteristics of many foodstuffs. The research in this area is important not only from the scientific point of view, but also from the view of impact's to the health as there is an increasing number of people suffering from allergies.

#### **4. Conclusion**

 A variety of allergens from different fruits were identified based on experimental immunology and molecular biology, that is, by sequencing, leading to gene and protein identification. Whereas allergens are typically described in certain plant species, each of them has a high degree of sequence identity to other proteins in their groups. Among the different fruit allergens, the pathogenesis-related (PR) proteins, classified into 17 families based on sequence, diverse structure, function and biological activity, and they are produced in response to different biotic and abiotic stresses. Allergens of individual plant food sources are very well described and structural details are known as well as the interaction with the immune system of patients. But at the level of regulation and expression of the genes themselves in plants, our knowledge is very limited for the known allergenic proteins. Basic genomic and transcriptomic analysis of the allergens will help to understand their natural genomic background in individual plant varieties and will lead to better personal allergy management in the future.

#### **Acknowledgements**

 This work was supported by the grants KEGA 007SPU-4/2017 and by The Danube strategy project DS-2016-0051.

### **Conflict of interest**

Authors declare no conflict of interest.

### **Thanks**

 The authors would like to thank Ing. Beáta Kováčová for her technical assistance and to colleagues from Department of Biochemistry and Biotechnology, namely Dr. Martin Vivodík for providing DNA of rye and buckwheat for this analysis.

### **Author details**

Jana Žiarovská1 \* and Lucia Zeleňáková2

1 Department of Genetics and Plant Breeding, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic

2 Department of Food Hygiene and Safety, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic

\*Address all correspondence to: jana.ziarovska@uniag.sk

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Application of Genomic Data for PCR Screening of Bet v 1 Conserved Sequence in Clinically… DOI: http://dx.doi.org/10.5772/intechopen.80312* 

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#### **Chapter 6**

## Insight into the Mechanism of Red Alga Reproduction. What Else Is Beyond Cystocarps Development?

*Pilar Garcia-Jimenez and Rafael R. Robaina* 

#### **Abstract**

 Volatile growth regulators play an important role in triggering aspects related to red seaweed reproduction. The last 10 years have brought clarification to how ethylene and methyl jasmonate work. Taking two reproductive stages of thalli of red seaweed—fertilised and fertile thalli—as benchmarks and a precise characterisation of the elicitation and disclosure periods of cystocarps, monitoring different gene expressions, namely candidate gene for reproduction and genes encoding proteins involved in biosynthesis pathways of both volatiles and reactive oxygen species, has enabled us to discern the differential behaviour of genes. These studies have also revealed that the volatile-mediated signal could affect cell wall loosening. All in all, studies have shown evidence of putative signalling pathways where volatile signal regulators form part of them at several levels, ranging from disclosure, development to the maturing of cystocarps. This signal information is crucial to determine the final response. The chapter also discusses whether signal transduction is related to different sensing for each volatile and whether this could be elicited in accordance with signal strength. This chapter compiles our current understanding of molecular mechanisms of algal reproduction and how volatile-mediated signals affect other developmental processes.

**Keywords:** ethylene, genes, methyl jasmonate, red seaweed, volatile growth regulators

#### **1. Introduction**

Carposporogenesis in red algae requires the disclosure and development of reproductive structures named cystocarps and cell wall weakness and also requires these reproductive structures to mature. Disclosure of cystocarps, in other words, the period in which the first cystocarps become visible, is elicited by external signals such as volatile growth regulators. On the other hand, controlling the elicitation period is essential for the proper development of cystocarps. If this period does not lead to the disclosure of cystocarps, cell wall loosening will not occur, and these structures will not mature either.

 Once the elicitation period occurs, cystocarp development begins with the weakening and relaxation of the cell wall. In Floridophyceae, the accepted view is that the cell wall is made of well-organised layers, whilst the intracellular matrix is comprised of less organised material. Some of components of the cell wall and matrix are sulphated galactans, which have a physiological significance that varies

#### *Systems Biology*

according to the different life stages of the macroalgae [1]. Despite the relationship between the loosening and weakening of the cell wall and the different life stages, the biochemical and molecular mechanisms have not been fully discovered. Evidence suggests that reactive oxygen species, under growth regulator control, are able to cleave cell wall polysaccharide, causing the wall to loosen [2, 3] during reproductive events in seaweed.

The maturity stages of reproductive structures in red algae are complex processes (**Figure 1**), highly co-ordinated and, to a large extent, quite difficult to determine. Unlike some seaweeds where different stages of development of cystocarps are evident and can be recognised [4], in others, the maturity stages are assumed to occur from the beginning of the discloser of the reproductive structures to thalli. In these cases, the maturity process differentiates between two kinds of thalli, the fertilised thalli and fertile thalli. The fertilised thalli are the ones that have both non-visible cystocarps and incipient visible cystocarps. Meanwhile, fertile thalli range from thalli with welldeveloped cystocarps to those that have fully mature cystocarps (**Figure 2A**).

Changes related to the maturing of thalli are favoured by volatile growth regulators, which also lead to both cystocarps dehiscence with a marked reduction of the maturity period, and the presence of different reproductive structures in the same individual [5]. Moreover, other evidence such as sudden losses of seaweed mats and alternating life cycles could also give cues on how volatile compounds act as a signal to trigger the reproductive process. Actually, seaweeds have a defined reproductive period and are able to discern between volatile signals. The latter leads to the presence of 'putative' volatile receptors although they are not yet known and only a proposed ethylene receptor in red algae has been reported [6].

 With this scenario, advances in gene studies have been made by combining different approaches—based on evidence of in vitro culture in the presence of volatile growth regulators and on algal physiology—and thus to decipher the network of interactions between different metabolic pathways that lead the transition from fertilised to fertile thalli. This path can lead to an understanding of a complex network of interacting genes and signal pathways that occurs. Hence one of the key questions is also to unveil how this process can be co-ordinated to work efficiently.

In recent years, great strides have been taken to gain understanding of molecular events in red seaweeds. These endeavours have allowed for a better understanding of the changes that occur during the transition from disclosure to the maturity of

#### **Figure 1.**

*Diagram of a tri-genetic life cycle in the red alga Grateloupia imbricata comprising the gametophyes (haploids), called carposporophyte, that develops on the female gametophyte after fertilisation, and the sporophyte (diploid).* 

*Insight into the Mechanism of Red Alga Reproduction. What Else Is Beyond Cystocarps… DOI: http://dx.doi.org/10.5772/intechopen.83353* 

#### **Figure 2.**

*(A) Schematic showing the timeline for the periods of disclosure and maturity of cystocarps and the corresponding fertilised thalli within cystocarps, ranging from non-visible to visible incipient and fertile thalli from well-developed to fully developed cystocarps. (B) Timeline of gene expression of ODC for fertilised and fertile thalli of Grateloupia imbricata after ethylene and methyl jasmonate treatment. The sloping lines represent significant increase or decrease in gene expression with respect to absolute values (copies μl −1 ). Horizontal line indicates no changes in gene expression relative to expression in control thalli. MeJa, methyl jasmonate.* 

 cystocarps in response to growth regulator. In this chapter we present our research output in the carragenophytic red seaweed model *Grateloupia imbricata*, compiling our current understanding of molecular mechanisms of algal reproduction and how a volatile-mediated signal can affect other developmental processes. This work does not forget to review other articles, but it does focus on what the state of the art is concerning red seaweed reproduction based on (1) candidate genes, (2) genes that encode cell wall weakness and reactive oxygen species and (3) genes that encode biosynthesis of volatile growth regulators such as ethylene and methyl jasmonate.

#### **2. Candidate gene of reproduction**

Growth, development and reproduction of multicellular organisms require precise and multifunctional cell–cell communication events. This is even more necessary in marine seaweed, where changes in irradiation, salinity and temperature, due to the tidal period, affect sporulation and mean that these organisms have to handle and adapt environmental signals in an extremely precise manner to survive. Taking this into consideration, it is easy to understand that algae make quick acclimation reversible acclimation—and adaptation to the marine environment and that the control of some of these vegetative and reproductive processes is particularly based on short-range signalling.

With this complex net of intervening factors, in order to interpret what is occurring with a particular event, the election of a candidate gene, which represents the manifestation of a trait such as the development and maturity of cystocarps, has provided insights into the carposporogenesis of red seaweeds.

Unlike unspecific genes that are overexpressed under a given condition and are assumed to be responsible for a particular event/trait/action [7–9], our candidate gene encodes the synthesis of the main protein ornithine decarboxylase (ODC, EC 4.1.1.17) responsible for the synthesis of polyamines and is related to the maturing process of cystocarps in seaweeds [10–12].

 The differential behaviour of this gene (*GiODC*) and its integration with volatiles contribute to it being chosen as a candidate gene for several reasons. First, the inhibition of ODC enzyme synthesis by the inhibitor DL-α-difluoromethylornithine (DFMO) implies the lowest levels of polyamines. This inhibition also halts the maturity of cystocarps and the eventual release of spores from cystocarps [11]. Second, the enzyme activity of ODC is related to the endogenous levels of polyamines. The reduction in ODC enzyme activity and polyamine levels are also related to the presence of cystocarps [10, 12]. Third, reactive oxygen species are released through polyamine catabolism pathways and are under the control of ODC. During cystocarp development, spermine is accumulated, favouring the process of development and maturity of the reproductive structure. When it exerts an inductive effect, polyamine oxidase enzyme activity increases as the spermine degrades [12]. Fourth, *GiODC* is expressed differently in both the fertile thalli (with cystocarps), than in infertile thalli (vegetative thalli), and in the apical part of fertile thalli, against the basal part of these thalli, as reported using -time quantitative PCR and in situ hybridisation techniques [13]. Fifth, sequencing the upstream region of *GiODC* revealed transcription factors involved in regulation by jasmonate (Myc2, Myc3 and Myc4) and ethylene (RAV, SMZ and Abi4). This means that there is a relationship between volatiles and *ODC* expression [14]. Sixth, monitoring *GiODC* gene expression after treatment with volatiles during the well-defined periods of elicitation and disclosure of cystocarps reveals differential behaviour of this gene, depending on the development and maturity of the cystocarps [14].

Putting all the data together suggests two important conclusions regarding the candidate gene: Expression is dependent on the existence of cystocarps and the kind of growth regulator used to elicit reproduction. Generally, down-expression of the gene candidate goes hand in hand with the presence of cystocarps and points to a quick transduction signal (**Figure 2B**) [15, 16]. Nonetheless, it is worth mentioning that there are two different gene expression patterns that occur when methyl jasmonate is used as an elicitor. Hence, in thalli without visible cystocarps, gene expression is upregulated due to methyl jasmonate signalling (fertilised thalli, **Figure 2B**). Moreover, in thalli containing fully mature cystocarps, other up-expressions are related to the stage of maturity of the cystocarp due to methyl jasmonate (fertile thalli, **Figure 2B**).

Far from being a mismatch for a candidate gene, it is understood that different signals are executed over the course of cystocarp development, and hence one can infer that thalli are able to discern between volatiles; they sense them in order to provide co-ordinated responses [14–16].

#### **3. Genes encoding proteins related to oxidative stress and softening of thalli**

 In most organisms, factors including drastic changes in temperature, irradiation and desiccation are stressful and potentially destructive. Nonetheless reproduction in algae is also highly regulated by temperature and tidal periods, which has an impact on processes such as sporulation. The generation of reactive oxygen species in turn is triggered by these environmental factors, as can be expected. To ameliorate this situation, organisms display various physiological responses which *Insight into the Mechanism of Red Alga Reproduction. What Else Is Beyond Cystocarps… DOI: http://dx.doi.org/10.5772/intechopen.83353* 

 are often being associated with an increase in the production of proteins that scavenge free radicals and reactive oxygen species (ROSs) [17, 18]. Unlike what has been well studied in higher plants, where stress proteins can be synthesised as a key survival strategy, we know that similar processes can occur, but it remains unclear whether stress proteins are metabolically biosynthesised or whether free radicals can be eliminated by chemical scavenging. Consideration also has to be given to the fact that certain red algae render methylate halides using methyltransferases that use S-adenosyl methionine (SAM, pivotal compound for the synthesis of ethylene and methyl jasmonate) as the methyl donor. Methylation of halides is a mechanism eliminating halide and sulphide ions, both of which are known to be phototoxic [19, 20].

Beyond this, seaweeds develop strategies to signal events related to growth and development, including the biosynthesis of volatiles. These volatile signals appear to crosstalk with other growth regulators such as polyamines [21]. As an example, polyamines, ethylene and methyl jasmonate share the same precursor—SAM—for these biosynthesis routes. Moreover, ROSs can be also released through metabolic pathways of growth regulators. The contribution of these signal pathways to growth and development is difficult to appraise as volatiles can have synergistic effects on one or more of the other pathways involved in seaweed reproduction, and this combination of all the pathways might give rise to several responses. Ethylene and methyl jasmonate provoke changes in the oxidation state of intermediates during synthesis. These include jasmonates, which are compounds, resulting from lipid oxidation of the cell membrane. In particular, methyl jasmonate is derived from linolenic acid, via lipoxygenase, in which the synthesis of methyl jasmonate activates the oxidative metabolism of polyunsaturated fatty acids, generating ROSs (in the form of O2, H2O2 or OH<sup>−</sup>) and oxidised derivatives of polyunsaturated fatty acids [22, 23]. Oxygenated volatile compounds have been shown to not necessarily involve photodamage of cell membranes. Meanwhile the reactivity of the ethylene double bond allows this olefin to be easily converted into a range of intermediates [24].

With this framework, ROSs also have the potential to interact with many cell components and can give rise to several physiological responses, such as when ROS acts as an important signal transduction molecule during growth [18]. Indeed, it has been inferred that ROSs play an important role in softening of thalli and therefore in the development of cystocarps in red seaweed. This is significantly important with the heat shock protein WD40 and cytochrome P450 which are responsible for reducing oxidative damage [25]. WD40 and cytochrome P450 are specifically related to ethylene and methyl jasmonate signalling [15, 16].

 Furthermore, what is striking is that genes that encode WD40 and cytochrome P450 mirror their expressions depending on whether they are elicited by ethylene or methyl jasmonate signals [15, 16]. The synchronised behaviour of these genes based on their expressions seems to determine close co-ordination due to the elicitor. Our results with *G. imbricata* suggest that the expression of one gene can become activated and repressed without the assistance of another one, but expression is also linked to different signals related to both cystocarp disclosure and development. In *G. imbricata*, this means that WD40 gene expression responds to the ethylene signal when cystocarps are still non-visible, whilst this gene expression increases in the presence of the first cystocarps after methyl jasmonate treatment (disclosure period). Otherwise cytochrome P450 is expressed in the presence of the first cystocarps (developing cystocarps) when they are treated with ethylene. Conversely, after the methyl jasmonate elicitor, cytochrome P450 expression responds when cystocarps are still invisible. In both cases, as the cystocarps mature, expression holds over time without any significant changes between thalli with well-developed cystocarps and fully developed cystocarps [15, 16] (**Figure 3**).

In addition, the ascorbate peroxidase gene, which encodes a protein involved in the response to oxidative stress [6, 15], is also associated with the disclosure and development of cystocarps rather than with their maturity process [16].

 Alternatively, polyamines, which are nonvolatile molecules but do have an important role in the process of maturing of the cystocarps, are synthesised through the candidate gene known as *ODC* [10, 14–16]. The synthesis of the polyamine precursor, putrescine, renders downstream spermidine and spermine due to the addition of one or two aminopropyl groups from decarboxylated SAM. Endogenous levels of these three polyamines—that is, putrescine, spermidine and spermine are balanced by amine oxidase and polyamine oxidase, whilest H2O2 is released as a by-product of this reaction.

Monitoring amine oxidase gene, whose gene expression was seen to depend on the disclosure and development period of cystocarps, but also that once cystocarps have developed, reported that this gene expression would help to maintain polyamines levels (**Figure 3**) [16].

 In short, our results confirm that genes encoding ROS proteins are related to physiological events. If we take the results as a whole, these behaviours of genes enable us to discern two action modes. Initially, WD40, cytochrome P450 and APX point to promoting the disclosure and development period of cystocarps, and they help to soften the thalli as up-expressions occurs. Meanwhile, amine oxidase expression shows a dual response. In other words, it helps cystocarp disclosure but it also balances ROS levels in order to fine-tune polyamine levels and prepare the thalli for the next time.

#### **Figure 3.**

*Timeline of gene expression encoding stress proteins (WD40, cytochrome P450 and amine oxidase) for fertilised and fertile thalli of Grateloupia imbricata after ethylene and methyl jasmonate treatment. The sloping lines represent significant increase or decrease in gene expression with respect to absolute values (copies μl −1 ). Horizontal line indicates no changes in gene expression relative to expression in control thalli. CytP450, cytochrome P450; AAO, amine oxidase; MeJa, methyl jasmonate.* 

#### **4. Genes encoding proteins involved in biosynthesis pathways of growth volatile regulators**

Despite the commercial importance of red seaweed, we still lack information on reproductive events if 'our' interest is to be able to control what happens over the course of the development and maturity processes of the reproductive structures and consequently manage to produce a large number of individuals.

Unlike the amount of information based mainly on next generation sequencing data, little progress has been made on the temporal control of genes, which affect growth and development. These aspects are of critical importance from the point of view of farming them. It is worth to highlight seaweeds that had received little attention worldwide to elucidate gene functions and to delve into the development and progress of functional genomic. Particularly in this section and as a practical goal, it is expected that molecular mechanisms related to volatile biosynthesis during carposporogenesis will provide tools for control and regulation of growth and developmental process in seaweeds. Thus, insight might allow to initiate a genetic programme for macroalgae which is economically valuable, increasing its viability and value.

 The molecular nature of the signal(s) that control development and maturity of cystocarps is unknown, although efforts have been made in recent years to accurately describe the elicitation and disclosure periods of cystocarps in the red alga *G. imbricata*. One of the most striking features is that alterations in gene expressions even start prior to the presence of visible cystocarps, which seems to suggest that communication through signal pathways is essential for the disclosure of cystocarps.

Our research team has focused on gene screening related to proteins specifically involved in biosynthesis pathways of volatile growth regulators instead of profiles of up- and downregulated genes reported in massive sequencing. Although it is obvious that any attempt is appropriate given the lack of molecular information in red seaweeds, we ought to bear in mind the existence of environmental acclimation of algae and the tremendous changes in the levels of expression of a large number of genes during the disclosure, development and maturity of cystocarps. Incidentally, we have to remember that factors such as salinity and sporulation are connected, and our aim is to be able to discern precisely what is happening.

Hence in order to gain a better and more accurate insight into the control mechanisms underlying the reproduction of red seaweeds, the monitoring of specific genes, that in turn are also related to growth regulators and their biosynthesis, has been successful (**Figure 4**). In particular, gene-encoding enzymes needed for the synthesis of ethylene, such as SAM synthase (SAMS) and ACC synthase (1-aminocyclopropane-1-carboxylate synthase), genes that encode proteins of polyamine metabolism (spermidine synthase (Spd synthase); amine oxidase), genes encoding proteins of methyl jasmonate synthesis, such as jasmonic acid carboxyl methyltransferase (JMT) and putative methyltransferase (MT); and a gene that encodes a transcription factor involved in controlling responses to stress, growth and development (MYB, [26]), have been monitored. These gene expressions have provided valuable information and helped to shed light on the complex process of red seaweed reproduction. As for genes related to ethylene biosynthesis, these are directly involved in cystocarp development, that is, SAMS, Spd synthase and ACC synthase. Otherwise, all genes studied in relation to methyl jasmonate are indiscriminately induced in the absence of cystocarps (**Figure 5**).

In general, we can indicate that methyl jasmonate and ethylene signalling occurs either immediately after the elicitation period or during the disclosure period, respectively (**Figure 5**). The time course of different gene expressions indicates a temporal regulation of algal reproduction. As part of this temporal regulation, the

#### **Figure 4.**

*Biosynthetic pathway for polyamines and connections with the pathways for the biosynthesis of ethylene and jasmonate. SAMS, S-adenosyl methionine synthase; d-SAM, decarboxylated SAM; MT, putative methyl transferase; JMT, jasmonic acid carboxyl methyl transferase.* 

#### **Figure 5.**

*Timeline of gene expression encoding biosynthesis proteins of ethylene (Spd synt, spermidine synthase; SAMS, S-adenosyl methionine synthase; ACCS, 1-aminocyclopropane-1-carboxylate synthase) and methyl jasmonate (JMT, jasmonic acid carboxyl methyl transferase; MT, methyl transferase) for fertilised and fertile thalli of Grateloupia imbricata after ethylene and methyl jasmonate treatment. The sloping lines represent significant increase or decrease in gene expression with respect to absolute values (copies μl −1 ). Horizontal line indicates no changes in gene expression relative to expression in control thalli.* 

differential gene expressions represent the ability of seaweeds to sense ethylene and methyl jasmonate separately [15, 16].

Signal transduction—like the presence of cystocarps—brings up the question of whether the sensing of both volatiles could be elicited in accordance with the signal strength. The latter is within the bounds of possibility since (i) ethylene, which is the smallest volatile molecule, can easily cross through cell membranes and (ii) the

*Insight into the Mechanism of Red Alga Reproduction. What Else Is Beyond Cystocarps… DOI: http://dx.doi.org/10.5772/intechopen.83353* 

hypothetical model of ethylene receptor for algae is a simpler structure than the one reported in higher plants. A priori, although both volatiles require membrane receptors, the fact is that the ethylene signal of the candidate gene elicited 12-fold the expression of methyl jasmonate despite the period where gene expression is reported [14]. This could be important for the fine regulation of disclosure and development of cystocarps.

 To make this more difficult, something else caught our attention. We have also wondered whether signal strength can be interpreted as a differential response between elicitor signal and signal transduction. Signal transduction is assumed to be the responsibility of a complex and integrated molecular network. The network for one or another volatile could overlap in such a manner that this overlapping simplifies signal channelling. Contrary to what some may think, we do not rule out separate signalling networks. Nevertheless, there could also be a signal output modulation 'mechanism' that regulates the disclosure and development of cystocarps [16]. We are a long way from knowing what is happening—in other words, the differential perception of volatiles, the separate and overlapping signal pathways and signal strength. Nonetheless, we realise that gene knockout studies will be advantageous to confirm these issues. Although we have accomplished the primary goal of revealing the molecular mechanisms underlying red seaweed reproduction, further studies are required to identify and explore other factors involved in the regulation of gene expression.

#### **5. Conclusions**

This chapter has summarised our insight into the complexity of gene regulation during red seaweed reproduction. There are grounds to believe that temporal patterns of gene expression are orchestrated under the control of volatile growth regulators signalling during the disclosure and development of cystocarps. Progress is being made in understanding how thalli transduce these volatile signals.

#### **Acknowledgements**

This work has been financed with funding from the Universidad de Las Palmas de Gran Canaria. Funding from Consejería de Economía, Industria, Comercio y Conocimiento del Gobierno de Canarias to PGJ is acknowledged (PROID2017010043 ACIISI; CEI2018-20 ULPGC).

#### **Conflict of interest**

The authors declare that there is no conflict of interest.

*Systems Biology* 

#### **Author details**

Pilar Garcia-Jimenez\* and Rafael R. Robaina Department of Biology, Faculty of Marine Sciences, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Canary Islands, Spain

\*Address all correspondence to: pilar.garcia@ulpgc.es

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Insight into the Mechanism of Red Alga Reproduction. What Else Is Beyond Cystocarps… DOI: http://dx.doi.org/10.5772/intechopen.83353* 

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*Systems Biology*

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[24] Garcia-Jimenez P, Brito-Romano O, Robaina RR. Production of volatiles by the red seaweed *Gelidium arbuscula* (Rhodophyta): Emission of ethylene and dimethyl sulfide. Journal of Phycology. 2013;**49**:661-669. DOI: 10.1111/jpy.12083

[25] Xu C, Min J. Structure and function of WD 40 domain proteins. Protein and

[26] Ambawat S, Sharma P, Yadav NR, Yadav RC. MYB transcription factor genes as regulators for plant responses: An overview. Physiology and Molecular Biology of Plants. 2013;**19**:307-321

2011;**33**(7):677-686

Cell. 2011;**2**:202-214

phospholipase, and oxylipin-production in the induced chemical defense of the red alga *Gracilaria chilensis* against epiphytes. Journal of Chemical Ecology.

[15] Garcia-Jimenez P, Montero-Fernandez M, Robaina RR. Molecular mechanisms underlying *Grateloupia*

[16] Garcia-Jimenez P, Montero-Fernandez M, Robaina RR. Analysis of ethylene-induced gene regulation during carposporogenesis in the red seaweed *Grateloupia imbricata* (Rhodophyta). Journal of Phycology. 2018;**54**:681-689. DOI: 10.1111/jpy.12762

carposporogenesis induced by methyl jasmonate. Journal of Phycology. 2017;**53**:1340-1344. DOI: 10.1111/

[17] Tripathy BC, Oelmüller R. Reactive oxygen species generation and signaling in plants. Plant Signaling and Behavior.

[18] Mittler R. ROS are good. Trends in Plant Science. 2017;**22**(1):11-19

LP. Methyl chloride transferase: A carbocation route for biosynthesis of halometabolites. Science.

[20] Roje S. S-Adenosyl-L-methionine:

Beyond the universal methyl group donor. Phytochemistry.

[21] Garcia Jimenez P, Robaina RR. Effects of ethylene on

tetrasporogenesis in *Pterocladiella capillacea* (Rhodophyta). Journal of Phycology. 2012;**48**:710-715. DOI: 10.1111/j.1529-8817.2012.01156.x

[22] Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R. Reactive oxygen species homeostasis and signaling during drought and salinity stresses. Plant, Cell and Environment. 2010;**33**:453-467

2012;**7**(12):1621-1633

[19] Wuosmaa AM, Hager

1990;**129**:160-162

2006;**67**:1686-1698

*imbricata* (Rhodophyta)

jpy.12594

**94**

### *Edited by Dimitrios Vlachakis*

Systems biology is the inevitable outcome of long years of knowledge acquisition and data accumulation. Te aim of systems biology is to integrate in a seamless way all existing knowledge in interconnected disciplines, stretching from modern biomedical research to physics, chemistry, and mathematics. Te main integration tool of such complex biomedical systems is via computational and mathematical modeling. In this direction, a series of state-of-the-art computer science techniques are used, namely, data mining and fusion, machine learning, and deep learning all under the prism of big data. All in all, systems biology is at the arrowhead of modern and state-of-theart biomedical research by atempting to address key biological questions describing holistically complex biological systems.

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