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Coffee is the second most traded global commodity next to oil and definitely the most

traded beverages of the world. Coffee, the brew prepared from the roasted seed of the

genus Coffea, also called the bean, has been historically described as the ‘Devil’s Drink’ .

Incidentally coffee was first popularized by the priest in Yemen because the neuro-

stimulatory effect it instigated proved beneficial for the long hours of traditional prayers

carried out by them. The name ‘Coffea’ originates from the Arabic derived term

‘Kahawa’ . By origin the native of the African subcontinent, coffee reached the Middle

East along with the captured African slaves and had been under restricted trade there due

to the capital it generated. For many years since then coffee has been a possession of

political and religious agendas between various nations. Today coffee is commercially

cultivated in 13 out of 25 biological diversity hotspots of the world, the leading producers

being Brazil. Coffee contributes to more than 75% of total Gross Domestic Product (GDP)

for 10% of the 72 cultivating countries and provides a source of income to 26 million

around the globe. Global coffee cultivation practice is shifting with the use of sun tolerant

varieties that drastically improve the yield (Gobbi, 2000). This may lead to serious failings

like deforestation, damage to bio-diversity, high inputs of pesticide and fertilizers and

soil/water deterioration (Elmqvist et al., 2000). India which is the sixth largest coffee

producer (311.52 thousand tonnes in the year 2013-2014) mainly relies on the traditional

shade grown coffee and is one of the countries known to grow coffee in the most eco-

friendly conditions (Marie-Vivien et al., 2014). In the crop year 2012/2013, the total

production was 145.1 million bags (one bag equivalent to 60Kgs) of which, Arabica coffee

constitutes 88.8 million bag and Robusta 56.2 million bags and the estimates at the end of

season for 2013/14 the production totalled 145.7 million bags (International Coffee

Organization statistics, www.ico.org).

Coffee research is concentrated in the areas of tissue culture, rust disease

resistance, berry borer resistance, aroma (trigonelline and reducing sugars), neuro-

stimulatory effects (caffeine), flavour (chlorogenic acids and caffeine) and transgenic

technology. Research on coffee has been very sporadic-the major work contributed by the

leads from Institutes like IRD and CIRAD from Montpellier, France and Cenicafe from

Colombia, and CIEFC from Portugal; however research activities has intensified off late

more rampantly in other countries as well. The complete sequencing of the coffee genome

and the first draft sequence published in ‘Science’ journal on September 4, 2014 issue,

brings in an enormous hope for rigorous activities to researchers and food scientists around

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the globe. As the famous Indian cafeteria ‘Cafe Day’ one liner goes “A lot can happen

over coffee”-A lot did happen over coffee. The infusion, coffee, has a great chemical and

biological complexity containing a mixture of more than a thousand chemical products.

Many bioactive components of coffee are broadly classified as the non-volatiles and the

volatiles. Among the purine alkaloids, caffeine is the most studied bioactive component

from coffee owing to its neuro-stimulatory properties. Caffeine (1,3,7- trimethylxanthine)

is found in the seeds and leaves of coffee, cocoa, mate, tea and cola. The biological role of

caffeine are mostly based on indirect evidences, nevertheless their pharmacological action

is quite well documented (Nehlig, 1999). Though moderate consumption of coffee has

been proposed to be beneficial for human health, excessive intake may lead to ailments

like anxiety, insomnia and high blood pressure, (James and Crosbie, 1987) owing to the

pharmacological properties of caffeine. The upper limit for caffeine consumption for a

healthy adult has been recommended to be below 400mg (2-3 cups of the brew each

measuring 150ml) (www.fda.org). This has lead to an increased demand for de-caffeinated

coffee.

Of the 124 species (Davis et al., 2011), only two species are commercially

important i.e., the highly regarded in terms of flavour, Coffea arabica (Arabica) and the

sturdy species with lower cup quality, Coffea canephora (Robusta) (Figure 1). C. arabica

is the only tetraploid species in the genus and presently accounts for around 60% of the

total produce. C. canephora are known to contain double the caffeine content compared to

C. arabica, the reason of which is unknown. It may be speculated that this difference in

caffeine content may be attributed to epigenetic mechanisms involving transcription

factors. Caffeine biosynthetic pathway has been a subject for studies in the past few

decades in both coffee and tea leading to a comprehensive picture of the biosynthetic

(reviewed by Ashihara et al., 2008), degradation (Mazzaferra et al., 1994) and the

dynamics of the biochemical pathway thus, culminating to the isolation, characterization,

transcript expression profiling (Ogawa et al., 2000, Mizuno et al., 2003a, Mizuno et al.,

2003b, Uefuji et al., 2003) and X-ray crystallographic structures of NMTs involved in the

core caffeine biosynthetic pathway (McCarthy and McCarthy, 2006). Xanthosine is the

committed precursor of caffeine biosynthetic pathway and it undergoes sequential

methylation at N7, N3 and N1 positions with a nucleosidase reaction prior to second

methylation step to produce caffeine. The methylation reactions are carried out by a large

family of plant proteins called as N- methyltransferases (NMTs). Alternatively, caffeine is

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synthesized by some minor routes like 7-methylxanthine → paraxanthine → caffeine, and

xanthine → 3-methylxanthine → theobromine → caffeine (Kato et. al, 1996).

Down-regulation of caffeine has been achieved through antisense and siRNA

technology, in both Coffea arabica and Coffea canephora, wherein silencing of one or

more of the three NMTs has been noticed (Ogita et al., 2004; Mohanan et al., 2013). In

addition to this, progress has been made to understand the upstream regions of coding part

of NMTs. The regulation of caffeine is far from understood, although it appears that light

and hormones regulate the pathway (Kurata et al., 1997, Aneja et al., 2001, Bailey et al.,

2005). These responses must act through modulating the expression/biological activation

of transcription factors leading to their interaction with the promoters of caffeine

biosynthetic genes.

Figure 1: The two cultivated species of Coffea genera. (A-D) The trees, flowers, fruits

and seed of Coffea canephora. (E-H) the tree, flower, fruits and seed of C. arabica.

PCR based genomic walking led to the isolation of 728bp 5' upstream promoter sequence

of the putative theobromine synthase gene (Satyanarayana et. al., 2005) at CSIR-Central

Food Technological Research Institute, Mysore, India. In silico analysis of the promoter

reveals various motifs for transcription factors involved in biotic and abiotic responses like

WRKY, and GT box respectively. WRKY family of transcription factors are the members

of a plant specific transcription factor family (Ülker and Somssich, 2004) that interact with

the W-box core elements (TGAC) on the promoters of responsive genes and regulate their

expression. All the WRKY proteins are Zinc finger type of transcription factors that carry

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one or two WRKY (W=Tryptophan, R-arginine, K= Lysine, Y=tyrosine) domains of 60

amino acid length with the signature amino acid sequences as WRKYGQK. Many studies

describe the functional role of WRKY transcription factors during plant development,

secondary metabolism and stress and disease response. However, no citings are available

for their involvement in caffeine biosynthesis regulation. Additionally, the characterization

of the promoter by serial deletions of the promoter and transcriptional silencing using

invert repeats of the promoter to reduce caffeine levels would provide the basic

understanding of the caffeine regulation in coffee.

Like any other secondary metabolite, caffeine content in coffee is also vulnerable

to various extrinsic and intrinisc factors. Recent studies have shown variations in caffeine

alkaloid content during the development and maturation of fruits and leaf (Perrois et al.,

2015), and also due to the influence of altitude at which coffee is grown (Sridevi and

Giridhar, 2014). Similarly, the influence of various biotic and abiotic stress on caffeine

content along with other quality attributing factors of coffee beans such as trigonelline,

niacin, cafestol and kahweol were reported (Sridevi and Giridhar, 2015). Although many

of the above studies indicated the role of regulation of gene in total caffeine accumulation,

the actual regulatory cis-elements of the promoter region and the trans-acting transcription

factor is yet to be identified. The role of caffeine as a protectant against pathogen/pest

attack (Uefuji et al., 2005; Kim and Sano 2008) and even in combating fungal infection

(Kim et al., 2011) in different genera other than Coffea is known. Even though caffeine

emerges as a component of plant defense mechanism, the role of key players of plant

defense response like salicylic acid (SA) and methyl jasmonate (MeJ) on expression of the

caffeine biosynthetic NMT genes is not well documented in coffee. Since NMT genes

appear to be regulated both developmentally as well as in response to stress signals as

evident by the studies on Coffea sp. (coffee), Camellia sp. (tea) and Theobroma sp.

(cocoa), the main question that arises is if these abiotic inductions are able to overcome the

development-induced transcription repression of the NMT genes or not. Hence, a more

comprehensive picture for transcriptional profile of NMT genes under various

environmental and biological stimuli is of prime importance for the groundwork of the

study on NMT gene regulation.

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Aims and Scope of the Present Investigation

The present study is the first detailed examination of transcriptional regulation of

caffeine biosynthetic pathway in coffee. Previous leads from Council for Scientific and

Industrial Research-Central Food Technological Research Institute, Mysore with the first

publication of sequence of promoter of theobromine synthase-like gene laid the foundation

for the present study. Due to annotation difficulties caused by involvement of highly

conserved multiple genes in the NMT family, an accurate phylogenetic analysis would be

required for assigning the function to the isolated theobromine synthase like gene. The in

vivo promoter activity by analysis of deletion constructs of the promoter fused upstream of

reporter gene in model plants would help identify the minimal promoter. The performance

of these deletion constructs in response to stimuli from light and defense would demarcate

the modular nature of the promoter and map regions that may be important for the binding

and activity of various transcription factors-the activators and the repressors. Furthermore,

transcriptional gene silencing using invert repeats against the promoter of the theobromine

synthase-like gene would essentially emphasize the contribution of this gene in the total

caffeine content, apart from the possibility of developing transgenic decaffeinated lines of

coffee. Finally, a comprehensive characterization of the WRKY superfamily of

transcription factors mined from C. canephora unigene database at the Solanaceae

Genomics Network (SGN) initiative, Cornell University, would serve as an essential

platform for analysis of defense related expression of caffeine in coffee plants.

Based on the literature reviewed above, the objectives of this thesis were laid down as:

1. Identification of different regulatory elements viz., light and defence that control

promoter of putative theobromine synthase involved in caffeine biosynthesis.

2. Differential expression of identified transcription factors viz., defence (WRKY)

and for light responsive, in C. canephora endosperms.

3. Transcriptional silencing of theobromine synthase gene using RNAi constructs

against putative theobromine synthase promoter in somatic embryos of Coffea sp.