Identification and Analysis of Jasmonate Pathway Genes in Coffea canephora (Robusta Coffee) by In Silico Approach.

BACKGROUND: Coffea canephora is the commonly cultivated coffee species in the world along with Coffea arabica. Different pests and pathogens affect the production and quality of the coffee. Jasmonic acid (JA) is a plant hormone which plays an important role in plants growth, development, and defense mechanisms, particularly against insect pests. The key enzymes involved in the production of JA are lipoxygenase, allene oxide synthase, allene oxide cyclase, and 12-oxo-phytodienoic reductase. There is no report on the genes involved in JA pathway in coffee plants.
OBJECTIVE: We made an attempt to identify and analyze the genes coding for these enzymes in C. canephora.
MATERIALS AND METHODS: First, protein sequences of jasmonate pathway genes from model plant Arabidopsis thaliana were identified in the National Center for Biotechnology Information (NCBI) database. These protein sequences were used to search the web-based database Coffee Genome Hub to identify homologous protein sequences in C. canephora genome using Basic Local Alignment Search Tool (BLAST).
RESULTS: Homologous protein sequences for key genes were identified in the C. canephora genome database. Protein sequences of the top matches were in turn used to search in NCBI database using BLAST tool to confirm the identity of the selected proteins and to identify closely related genes in species. The protein sequences from C. canephora database and the top matches in NCBI were aligned, and phylogenetic trees were constructed using MEGA6 software and identified the genetic distance of the respective genes. The study identified the four key genes of JA pathway in C. canephora, confirming the conserved nature of the pathway in coffee. The study expected to be useful to further explore the defense mechanisms of coffee plants.
CONCLUSION: JA is a plant hormone that plays an important role in plant defense against insect pests. Genes coding for the 4 key enzymes involved in the production of JA viz., LOX, AOS, AOC, and OPR are identified in C. canephora (robusta coffee) by bioinformatic approaches confirming the conserved nature of the pathway in coffee. The findings are useful to understand the defense mechanisms of C. canephora and coffee breeding in the long run.
SUMMARY: JA is a plant hormone that plays an important role in plant defense against insect pests. Genes coding for the 4 key enzymes involved in the production of JA viz., LOX, AOS, AOC and OPR were identified and analyzed in C. canephora (robusta coffee) by in silico approach. The study has confirmed the conserved nature of JA pathway in coffee; the findings are useful to further explore the defense mechanisms of coffee plants. Abbreviations used:C. canephora: Coffea canephora; C. arabica: Coffea arabica; JA: Jasmonic acid; CGH: Coffee Genome Hub; NCBI: National Centre for Biotechnology Information; BLAST: Basic Local Alignment Search Tool; A. thaliana: Arabidopsis thaliana; LOX: Lipoxygenase, AOS: Allene oxide synthase; AOC: Allene oxide cyclase; OPR: 12 oxo phytodienoic reductase.

INTRODUCTION

Jasmonic acid (JA) and its derivatives jasmonates (JAs) play a key role in plant metabolic processes and signaling systems when they are in stress and injured from pests.[1234] JAs, also called as oxylipins, are derived from fatty acid α-linolenic acid through the formation of different intermediates, i.e., 13-hydroperoxy-9,11,15-octadecatrienoic acid by 13-lipoxygenase (LOX), 12-oxo-phytodienoic acid (OPDA) by allene oxide synthase (AOS) and allene oxide cyclase (AOC) in cytoplasm, and finally JA by OPDA reductase (OPR) through 3-oxo-2 (2’[Z]-pentenyl) cyclopentane-1-octanoic acid by β-oxidation in peroxisomes [Figure 1].[456] The synthesis and signaling process of JA and JAs what we presently know is from studies on Arabidopsis thaliana and Solanum lycopersicum.[789] Still, in many plant species including coffee, the jasmonate pathway is yet to be studied in detail. These plants have to be studied to know if they possess exactly the same biosynthetic pathway comprising similar enzymes that perform the same biological role in their metabolism as in Arabidopsis and Solanum.

Figure 1

Jasmonic acid is synthesized from fatty acid linolenic acid. Lipoxygenase, allene oxide synthase, allene oxide cyclase are the key enzymes of jasmonate biosynthesis in chloroplast, and they form oxo-phytodienoic acid. Oxo-phytodienoic acid is transported to peroxisome. Reduction of cyclopentanone ring of oxo-phytodienoic acid is catalyzed by peroxisomal oxo-phytodienoic acid reductase enzyme. Three cycles of β-oxidation occur to give finally jasmonic acid

Coffee is one of the most important agricultural commodities; nearly 124 species are identified and exist around the world.[10] However, only two species are considered commercially important, i.e., Coffea arabica L and Coffea canephora P, of which C. arabica is known for better cup quality compared to C. canephora. Between the two species, arabica is more sensitive to pathogens and pests (fungi, nematodes, and insects).[11] In recent years, many coffee growers are shifting to robusta coffee cultivation due to high production costs, labor scarcity, and disease and pest management associated with arabica cultivation.

The objective of the study was to identify key genes of JA biosynthesis pathway in C. canephora, which is one of the parents of the tetraploid arabica coffee along with Coffea eugenioides. Recently, C. canephora genome is sequenced producing a high-quality draft genome of the species.[12] The complete genome sequences of C. canephora are available in the web-based database of Coffee Genome Hub (http://coffee-genome.org/). The database served as a very useful resource in this study.

MATERIALS AND METHODS

Databases

GenBank Database of National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) and database of Coffee Genome Hub (http://coffee-genome.org/) were used to identify the presence of proteins of the key enzymes involved in JA synthesis.

Software

Molecular evolutionary genetic analysis software MEGA6 (http://mega6.software.informer.com/) was used for sequence alignment and construction of phylogenetic trees.

Identification of protein sequence of key genes of Coffea canephora involved in jasmonic acid biosynthesis

Protein sequence of different genes involved in the JA synthesis was taken from the model plant A. thaliana using NCBI database. Those sequences were used for Basic Local Alignment Search Tool (BLAST) search[13] and the Coffee Genome Hub database to identify the respective gene sequences in C. canephora. Among the matches in C. canephora, top match for each gene was taken and used for BLAST search against NCBI database. The top matches for C. canephora proteins involved in the JA pathway obtained in NCBI BLAST search were used for sequence alignment to obtain phylogeny trees using MEGA6 software.[14]

Construction of phylogenetic trees

Around 14 top matches in NCBI database for each protein involved in the JA pathway and 1 top match from coffee genome database were taken from the BLAST results and transferred the sequence data to MEGA ALIGN tool in MEGA6 software. Using CLUSTALW alignment, the obtained sequences were aligned and by neighbor-joining method with bootstrap value 1000, the phylogenic trees were constructed.[15]

RESULTS AND DISCUSSION

The main enzymes that play a key role in the biosynthesis of JA are LOX, AOS, AOC, and OPR[1] [Figure 1]. The enzyme LOX is involved in the production of the intermediate compound 13-HPOT by adding oxygen to α-linolenic acid at either C9 or C13 position, with 9S- or 13S-hydroperoxides.[161718] AOS and AOC are involved in the formation of 12-OPDA through unstable compound, an allene oxide.[19] It is encoded by single gene in Arabidopsis,[2021] two genes in tomato.[22] OPR belongs to a small family of related flavin-dependent oxidoreductases forms the JA through β-oxidation in peroxisome.[462324] The proteins of these enzymes were taken from model plant A. thaliana in NCBI database. Selected protein sequences of A. thaliana were used to identify homologous protein sequences in C. canephora genome from Coffee Genome Hub database using BLAST tool. We identified the matching protein sequences related to LOX, AOS, AOC, and OPR enzymes with similarities ranging from 2 to 17 [Table 1].

Table 1

Arabidopsis thaliana protein sequences of key genes involved in jasmonic acid biosynthesis from National Center for Biotechnology Information used as query and Coffea canephora matches obtained in coffee genome hub database

The same sequences were cross-checked again from coffee genome database with NCBI database and identified as unnamed protein products related to LOX, AOS, AOC, and OPR enzymes in C. canephora. The maximum number of similarities obtained in coffee genome database was 17 for OPR [Table 2], 7 for AOS [Table 3] and 11 for LOX [Table 4]. The minimum number of similarities obtained was 2 for AOC [Table 5]. Top one similarity for each enzyme was taken as main source to confirm with NCBI by doing BLAST. Matches in NCBI database for each gene were taken for phylogenetic tree construction. The evolutionary history was inferred using the neighbor-joining method.[25] The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.[26] The evolutionary distances were computed using the Poisson correction method[27] and are in the units of the number of amino acid substitutions per site. The analysis involved 15 amino acid sequences. Evolutionary analyses were conducted in MEGA6.[28] LOX, AOS, AOC, and OPR trees mainly show two broad clusters. Each cluster is further divided into subclusters.

Table 2

Matches obtained for 12-oxophytodienoate reductase in Coffea canephora from coffee genome database. Arabidopsis thaliana protein sequence CAB66143.1 was used as query

Table 3

Matches obtained for allene oxide synthase in Coffea canephora from coffee genome hub database. Arabidopsis thaliana protein sequence NP_199079.1was used as query

Table 4

Matches obtained for lipoxygenase in Coffea canephora genome from coffee genome hub database. Arabidopsis thaliana protein sequence AAF97315.1 was used as query

Table 5

Matches obtained for allene oxide cyclase in Coffea canephora from coffee genome hub database. Arabidopsis thaliana protein sequence CAC83763.1 was used as query

The LOX tree mainly shows two broad clusters. Each cluster is further divided into subclusters. For LOX enzyme protein, one subcluster unnamed protein product from C. canephora and 13s-LOX (Cc00_g30760) from coffee genome was showing bootstrap value 100. In addition to this, Theobroma cacao and Gossypium hirsutum also showed bootstrap value 100 [Figure 2]. In AOS, one subcluster unnamed protein product from C. canephora and AOS (Cc10_g03580) from coffee genome was showing bootstrap value 100; in addition to this, T. cacao, G. hirsutum, Glycine max, and Lotus japonicus also showing bootstrap value 100 and Catharanthus roseus 99 [Figure 3]. In AOC, one subcluster unnamed protein product from C. canephora and AOC 4 (Cc07_g09040) from coffee genome was showing bootstrap value 100. Nicotiana tabacum and Petunia x hybrida were showing 90 and S. lycopersicum was showing bootstrap value 100. In addition to this, T. cacao and G. hirsutum are showing bootstrap value 99 [Figure 4]. OPR tree also mainly shows two broad clusters. Each cluster is further divided into subclusters. In subcluster I, unnamed protein product from C. canephora and 12-OPR (Cc06_g12110) which was taken from coffee genome was showing bootstrap value 100. In addition to this, T. cacao and G. hirsutum also showing bootstrap value 100; Nicotiana tabacum and S. lycopersicum showing bootstrap value 99 [Figure 5].

Figure 2

Phylogenetic tree for lipoxygenase enzyme protein

Figure 3

Phylogenetic tree for allene oxide synthase enzyme protein

Figure 4

Phylogenetic tree for allene oxide cyclase enzyme protein

Figure 5

Phylogenetic tree for the oxo-phytodienoic acid reductase enzyme protein

The present study confirms the presence of genes involved in the synthesis of JA pathway in C. canephora, and further studies are needed to know the complete mechanism of these genes during signal transduction in resistance from pests compared to other coffee species.

CONCLUSION

JA pathway is reported to have mainly LOX, AOS, AOC, and OPR enzymes occurs in chloroplast and peroxisome, but there was no report of the presence of these enzymes in coffee plants. From the above study, we are concluding that the JA synthesis enzymes present in the pest-resistant coffee plant C. canephora and identified the close relatives of each gene in the NCBI database related to coffee plant. This study will be useful to understand the resistance mechanism in C. canephora, and this study is helpful to increase the resistance in other coffee plants such as C. arabica by artificial JA application in their cultivation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.