CHAPTER 2 Review of Literature -...

CHAPTER 2

Review of Literature

Chapter 2 Review of Literature

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2.1. Sugarcane

Sugarcane is one of the potential grass which contributes to the world‟s major source

of sucrose about 75 % with value more than US$ 150 billion per year. Native to warm

temperate to tropical regions of Asia, they have stout, jointed, fibrous stalks that are

rich in sugar, and measure two to six meters (six to nineteen feet) tall. All sugarcane

species are inter-breed and the major commercial cultivars are complex hybrids

(Mukherjee, 1957; Daniels and Roach 1987).

Cultivation of sugarcane

The grass family (Poaceae) is an economically important seed plant family that

includes maize, wheat, rice, and sorghum and many forage crops. Sugarcane

(Saccharum spp. hybrid) is a C4 grass belongs to genus Saccharum of the family

Poaceae with complex polyploidy genome as a polyploidy and aneuploid. The

scientific classification is as given in Table 2.1 as per the Integrated Taxonmic

Integrated System (http://www.itis.gov/) upated as on April 2015 The stems of the

sugarcane grow into thin, tall cane stalk which approximately comprise 80 % of the

entire plant. The mature cane characteristically composed of 12–16 % fiber and

soluble sugars, 2-3 % of the non-sugars and the higher content of water 60-70 %. The

crop is seen to be very responsive towards the climate change, soil type, irrigation

facilities, fertilizer applications, biotic and abiotic stresses, and varied harvest period.

thus, the average yield of the cane is roughly around 60-70 tonnes per hectare per


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year. the yields of the cane differ depending on the crop management approach and

the comprehension of the sugarcane cultivation. The cane is moreover considered as a

cash crop but is eaqually regarded as a livestock fodder also.

Table: 2.1 Scientific classification of sugarcane from Integrated Taxonmic

Integrated System updated as on April 2015

Kingdom Plantae plants

Subkingdom Viridiplantae

Infrakingdom Streptophyta land plants

Superdivision Embryophyta

Division Tracheophyta vascular plants

Subdivision Spermatophytina spermatophytes

Class Magnoliopsida

Superorder Lilianae monocotyledons

Order Poales

Family Poaceae grasses

Subfamily Panicoideae

Tribe Andropogoneae

Genus Saccharum L. complex sugarcane

2.2 Sugarcane industry

Apart from the traditional use as a source of sugar, sugarcane is fast becoming a

source for ethanol and biomass production as an alternative energy source to the non-

renewable resources in many counties as a part to build up their economy from

several decades. It efficiently grows vegetative as well as reproductive way in the

tropical and sub-tropical regions of more than 90 countries with area under cultivation

close to 20 millions of hectares (FAO 2008; http://faostat.fao.org/;

http://www.illovo.co.za/worldofsugar). The Indian sugar industry play an important

role in global market being the world‟s second largest producer after Brazil,

producing nearly 15 % sugar and 25 % sugarcane per annum under a wide range of

agro-climatic conditions, both in tropical and subtropical regions. In 2010, Nigeria

was the 2nd

largest producer of sugarcane in West Africa after Ivory Coast and the 19th

in Africa. Currently, the industry produces around 300-350 million tones Mt cane, 20-

http://www.illovo.co.za/worldofsugar


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22 Mt white sugar and 6-8 Mt jaggery and khandsari to meet the domestic

consumption of sweeteners ((FAOSTAT, 2012; Solomon, 2011). This view

accelerates the sugarcane production in most of the developing and developed

countries globally (Alexander, 1986). The Brazilian industry leads in the sugarcane

processing and allied product generation (Manners and Casu 2011; Matsuoka et al

2009). It is one of the world's biggest producer of fuel ethanol (6921 million gallons

in 2010 from sugar cane) and the world's biggest exporter of fuel ethanol without any

deforestation which potentially meets 14 % of the biofuel (Somerville et. al. 2010).

The year 2011 data showed that the Brazil is leading supplier of renewable sources

with 44.1 percent of the energy matrix in 2011 whereas it represents 8 percent of the

total for the Economic Cooperation and Development (OCED) countries (European

biofuel technology platform http://www.biofuelstp.eu/s_america.html; Nass et. al.

2007; Goldemberg, 2008).

It has been stated that the developing counties consume roughly 26 % of the world‟s

energy, yet represent about 6 % of the world‟s population (Louime et al. 2008;

Shrivastava et. al. 2014). Worldwide sugarcane researchers are trying to enhance the

potential of sugarcane that can make a substitute to the non-renewable nature of fossil

fuels. The progress of economically important sugarcane research emerges from the

conventional breeding, genome understanding, gene discovery and molecular

breeding. Sugarcane improvement, from selection of existing variation in pre-historic

time to the current bi/multi-parental crossing and subsequent use of non-conventional

techniques, has concentrated mostly on improving the yield and sugar content.

2.3 Abiotic stress

The external altered conditions surrounding the plants have a distressing impact on

the plant growth and its yield. Nonetheless, the consequences of these altered

conditions on the plants are majorly evaluated under the controlled growth conditions

in vitro. In constrast, the environmental conditions are seem to be very varied and can

engage a combination of the stresses such as drought and cold, heat and drought,

salinity and heat or a biotic and abiotic combined stress (Suzuki et. al. 2014). Studies

are being progressed to comprehend the response of these types of the combined


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stresses in the plants. Due to the high degree of concurrent occurrence of the stresses

the intracacies in the plant response increases. The cause for these intracacies thus

cannot be traced easily and the plant cannot survive the stress condition. Due to the

stress effect collision of the plant growth and its physiological condition is resulted.

The chief abiotic stresses widely studied which affect the crops in the field consist of

drought, salinity, heat, cold, chilling, freezing, nutrient, high light intensity, ozone

(O3) and anaerobic stresses (Wang et. al. 2003, Chaves and Oliveira 2004; Agarwal

and Grover 2006; Nakashima and Shinozaki 2006; Hirel et. al. 2007; Serres and

Voesenek 2008, Chinnusamy and Zhu 2009; Mittler and Blumwald 2010). Research

carried out on the drought environmental stress has revealed crop growth and yield to

be limited and many physiological changes occur which might affect the crop quality.

(Rizhsky et. al. 2002, 2004; Mittler, 2006; Prasad et. al. 2011; Vile et. al. 2012). Thus,

to fulfil the needs of the gobal food demand of the ever increasing population, it has

become crucial to upgrade the crops with the superior tolerance to the abiotic stresses

such as drought, salt, heat, freezing and their combinations.

In a latest study, the sugarcane drought-tolerant and sensitive cultivars were compared

for their growth and the physiological characters when subjected to drought, cold

stress, and their combination (Sales et. al. 2013). It was experiential that the sensitive

cultivars showed reduction in the biomass in constrast to the tolerant ones. Also the

effect of the drought and heat combination showed higher decrease in the biomass

along with the photosynthetic rate being severely affected than the single effect of

drought in sugarcane and as well as barley crops (Rollins et. al. 2013; Sales et. al.

2013).

Researchers predict the antioxidant defense machinery to be one of the key pathways

which appears to trigger the tolerance of the plants to the stress conditions. It was

directed that the association of the higher antioxidant capability or low reactive oxgen

species buildup aid the tolerance of plants towards the stress conditions (Demirevska

et. al. 2010; Ahmed et. al. 2013; Iyer et. al. 2013; Perez-Lopez et. al. 2013; Sales et.

al. 2013; Rivero et. al. 2014). The plant is seen to follow two types of mechanisms to

combat the adverse condition. The plants pursue the stress tolerance path or the stress

avoidance path. The stress avoidance path of the plants is in form of seeds before the


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drought condition prevails, or by adaptation in the plant architecture, or by

morphological adaptations of developing specialized leaf sufaces to decerese the

transpiration rate, sunken stomata, increase in the root length and density to use water

more efficiently. Also the intricate trait of tolerating the stress emerges to be the

consequence of the co-ordination of biochemical and physiological alterations at the

cellular and molecular level by accumulating the various osmolytes. Late

embryogenesis-abundant proteins coupled with an efficient antioxidant system (Shao

et. al. 2008; 2009).

2.4 Advancements to direct the abiotic stress condition in crops

To meet the demands, the development of new sugarcane varieties for the improve

productivity, tolerance to biotic and abiotic stresses, nutrient management, and

improved sugar recovery are some of the challenges need to be overcomed.

Drought and salinity are the most severe limitations on the yield of sugarcane. Indian

sugarcane productions are badly affected by water scarcity because of the poor

irrigation facilities and hence it creates stress to the plant. This stress induces various

biochemical and physiological responses in plants as a survival mechanism (Seki et.

al. 2003; Patade et. al. 2011). Various strategies are developed in plants under stress

conditions. In terms of biochemical responses, stress induces accumulation of

functioning solutes, such as proline, sugar, sugar alcohol and betaine in plants

(Rhodes and Hanson 1993; Ingram and Bartels 1996). These compounds facilitate

adaptation of plants under severe circumstances. Regarding the genetic responses, a

variety of genes have been reported to be induced by stress in various plant species.

The function of the expressed proteins has

been predicted to play a vital role in the adaptive response to stress (Ingram and

Bartels 1996; Bray, 1997) Drought and salinity in the root zones of sugarcane

decreases the sucrose yield, through its effect on both biomass and juice quality. The

complexity and polygenic nature of stress tolerance has further limited the efforts to

develop the tolerant crop varieties through conventional breeding practices. In this

regard, biotechnological approaches including somaclonal variation, in vitro

mutagenesis and selection are being applied for the isolation of agronomically useful


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mutants (Jain et. al. 2005). Agronomically improved sugarcane varieties endowed

with tolerance to biotic and abiotic stresses are highly beneficial, as unfavorable

environmental factors can challenge cultivation and crop productivity. Although crops

tolerant to biotic and abiotic stresses have been selected by traditional breeding

programs, speeding up the pace is essential in developing improved varieties (Seki et.

al. 2001).

Stress conditions have been one of the most critical state in agriculture and many

efforts have been made to improve crop productivity under such conditions. Many

breeders have employed natural breeding selection as the criteria for improving the

tolerance of this crop (Tuberosa and Salvi 2006). But due to attack of the abiotic and

biotic stresses in the chickpea crop is devastated against the breeder‟s efforts. This

natural selection mechanism favours the adaptation and survival whilst the breeding

programme has directed towards the increased economic yield of this chickpea crop

(Jha et. al. 2014). Many physiological studies have categorize the traits associated

with the adaption of the crop to the drought stresses such as small plant size, reduced

leaf area, early maturity and prolonged stomatal closure lead to a reduced total

seasonal evapotranspiration, late heading and flowering and to a reduced yield

potential (van Ginkel et. al. 1998; Karamanos and Papatheohari, 1999). The

conventional approach of the breeding and selection has achieved limited success for

release of abiotic and biotic stress tolerance crop (Richards, 2006). Even the wild

sugarcane varieties were considered to be used for transformation experiments with

respect to the stress tolerance trait. But the intricacy and size of the sugarcane genome

has been a major restraint in genetic improvement and gave very stumpy results. In

such complex polyploidy crop no single technique is found to be better for

confirmation of polygenic and phenotypic characters, hence other approaches such as

metabolomics and proteomics open up the complex network association through in

silico methodology and laboratory work for particular traits.

Thus, the introduction of the molecular approaches for gene transfer was considered

to be the revolutionary aspect. Sugarcane genomics is also focusing in isolation of

novel transcription factors responsible for stress tolerant conditions and studies in

their functional characterization.


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2.5 Plant transcription factors and abiotic stress

Transcription factors (TFs) are the key regulatory proteins that augment or repress the

transcriptional rate of their target genes by binding to specific promoter regions. They

are important regulators of gene expression mainly composed of four discrete

domains, DNA binding domain, nuclear localization signal (NLS), transcription

activation domain, and oligomerization site. These function together to regulate

numerous physiological and biochemical processes by modifying the rate of

transcription initiation of the target gene (Du et. al. 2009). It is assumed that the TFs

act together with the other transcriptional regulators which include the chromatin

remodeling and modifying proteins so as to employ or obstruct the RNA polymerases

to bind the DNA template (Udvardi et. al. 2007). The plant genomes consign roughly

7 % of their coding sequence to the TFs, which proves the intricacy of transcriptional

regulation (Udvardi et. al. 2007). The TFs interact with cis-elements and other

proteins which activate or repress the transcription of target genes by their functional

domains. Moreover, in the promoter regions of several stress-related genes and thus

up-regulate the expression of many downstream genes result in imparting abiotic

stress tolerance (Agarwal and Jha 2010). They play fundamental roles in almost all

biological processes (development, growth and response to environmental stress

conditions) and are assumed to participate immensely during the evolution of plant

system. In Arabidopsis thaliana genome approximately 1500 TFs are described which

are considered to be concerned for stress responsive gene expression (Riechmann

et. al. 2000). The transcriptome data in Arabidopsis and in other several plants imply

that there are number of pathways that work independently to respond to the

environmental stresses (both ABA dependent and ABA independent) thus, suggesting

that stress tolerance or the susceptibility is controlled at the transcriptional level by an

extremely obscure gene regulatory network. (Fowler and Thomashow, 2002;

Umezawa et. al. 2006).

Based on the structural characteristics their DNA-binding domain are helix–loop–

helix, zinc finger, helix–turn–helix, leucine zipper, scissors, MADS cassette, etc.

These TFs can be classified into different families viz; bZIP, MYB, WRKY,

AP2/DREBP and some zinc finger like proteins. The central regulator of abiotic stress


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particularly drought resistance in plants is seen to coordinate a multifaceted gene

regulatory network which enables the plants to cope decreased water availability

circumstance (Cutler et. al. 2010; Kim et. al. 2010).

The graphic depiction of cis-acting elements of the transcriptional regulatory

networks involved in abiotic stresses with the respective transcription factors are

shown in figure 2.1. The transcriptional regulatory network system of the cis-acting

elements and the transcription factors which involves in the ABA and abiotic stress

responsive gene expression contain the cis-regulatory elements of the promoters of

the stress-induced genes such as MYC recognition sequence (MYCRS; CANNTG)

and MYB recognition sequence (MYBRS; C/TAACNA/G), DRE/CRT (A/GCCGAC),

ABRE (PyACGTGGC), which are seen to regulated by diverse upstream

transcriptional factors (Fig. 2) (Zhu, 2001; Mahajan and Tuteja 2005; Tuteja 2007).

The ABA-dependent stress signaling pathways are seen to activate the basic leucine

zipper transcription factors called the AREB, which binds to ABRE element to induce

the stress responsive gene (RD29B). In Arabidopsis, it is reported that two ABRE

motifs are involved in the regulation of ABA-responsive expression of the RD29B

gene, which encodes a LEA-like (late embryogenesis abundant) protein (Hundertmark

and Hincha 2008; Yoshida et. al. 2010). Wide range of studies have revealed the

potential role of LEA proteins in the stress tolerance. Their role in the protein

protection upon water deficit was illustrated by in vitro experiments, where different

LEA proteins were able to prevent the inactivation of enzymes such as lactate

dehydrogenase or malate dehydrogenase upon different dehydration levels (Goyal et.

al. 2005; Reyes et. al. 2008). Transcription factors such as DREB2A and DREB2B

trans-activate the DRE cis-element of osmotic stress genes and are thus concerned in

retaining the osmotic stability of the cell (Mahajan and Tuteja 2005). Some of the

genes similar to RD22 lack the classic CRT/DRE elements in their promoter region

which signify their regulation to be occurred by some other mechanism. This RD22

gene protein have a homology to an unidentified seed protein. The drought inducible

expression of DREB1D is seen to be regulated by ABA- dependent pathways,

demonstrating that DREB1D protein might be involved in the function of slow

response to drought that depends upon the buildup of ABA (Fig. 2.1).


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Fig. 2.1 Transcriptional regulatory network of cis-acting elements

ABA-dependent and ABA-independent transcription factors involved in the abiotic stresses which

control the stress-inducible gene expression are shown in ovals. ABA: abscisic acid; ABRE:

ABA-responsive element; CBF: C-repeat-binding factor; COR: cold regulated genes; CRT: C-repeat;

DRE: dehydration-responsive element; DREB: DRE-binding protein; ERDearly responsive to

dehydration, MYB: myeloblastosis; MYBRS: MYB-recognition sequence; MYC: myelocytomato-sis;

MYCRS: MYC-recognition sequence; NACR: NAC-recognition site; RD: genes responsive to

dehydration; ZF-HD: zinc-finger homeodomain. (Modified from Tuteja et al 2007 and reference

therein)

The transcription factors MYC/MYB, RD22BP1 and AtMYB2, could bind MYC and

MYB recognition sequence elements, respectively and assist in establishment of the

RD22 gene (Fig. 2.1). These MYC and MYB proteins are identified and synthesized

only after endogenous levels of ABA build up, consequently suggesting that their


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functions are in a later stages of the stress responses (Mahajan and Tuteja 2005).

2.6 Plant MYB transcription factors

The MYC/MYB families and their proteins are unanimously derived from both plants

and animals and are recognized for their varied functions. Wide existence of the MYB

transcription factor genes specify their ancient evolution. The first MYB gene

identified was the v-MYB gene from Avian Myeloblastosis Virus (AMV)

(Klempnauer et.al. 1982). Furthermore, the first plant gene encoding MYB

transcription factor, COLORED1 (C1), was isolated from grass family, Zea mays, and

is found to encode a c-MYB protein domain involved in anthocyanin biosynthesis in

aleurone (Paz-Ares et. al. 1987). Three v-MYB-related genes, c-MYB, a-MYB, and b-

MYB, were subsequently found in many vertebrates, and were thought to be involved

in the regulation of cell proliferation, differentiation, and apoptosis (Weston, 1998) in

Drosophila and Dictyostelium. Homologous MYB genes were also identified in

insects, fungi, and slime molds (Lipsick, 1996). Interestingly, the numbers of MYB

genes in plants are remarkably higher than those in fungi or animals (Riechmann et.

al. 2000). All of the three-repeat of the MYB proteins seem to appear to bind to the

same core DNA sequence motif and fall into

distinct families.

These MYB proteins are divided into different classes on the number of adjacent

repeats present (one, two, three or four; Fig. 2.2). Most of the plant MYB genes

encode the R2R3-MYB type of class and are thought to be evolved from 3R-MYB gene

ancestor with two adjacent repeats by loss of sequences encoding the R1 repeat (Fig.

2.2).

These R2R3-MYB transcription factors have a modular structure, with an N terminal

DNA-binding domain, which is the MYB domain, and an activation or repression

domain usually located at the C terminus. In higher plants, this protein family is

extraordinarily amplified (Romero et. al. 1998; Rabinowicz et. al. 1999; Stracke et. al.

2001) and explored in the study of MYB transcription factors for understanding its

role and functioning pathway. The three repeats of the prototypic c-MYB protein are

denoted as R1, R2 and R3. The three repeats of c-MYB. All four repeat classes are


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Fig. 2.2: Schematic representation of plant MYB transcription factor classes

A) Depicts the different plant MYB protein classes depending on the number of adjacent MYB repeats

1) represents the 1R-MYB, MYB-related and other MYB types 2) represents the 3R-MYB type and 3)

represents the 4R-MYB type.

B) Depicts the primary and secondary structures of a typical R2R3-MYB protein. H- helix; T- turn; W-

tryptophan; X- amino acid. (Modified from Dubos et. al. 2010, and references therein).

found in plants, representing the taxon with the highest diversity of MYB proteins.

The 4R-MYB group is the smallest class having four R1/R2-like repeats and is found

to be encoded in several plant genomes (Fig. 2.2 A3). The class two contains

R1R2R3-MYB (3R-MYB) with three adjacent repeats are typically encoded by five

genes in higher plant genomes and play role in cell cycle control (Haga et. al. 2007;

Du et. al. 2012) (Fig. 2.2 A2). The third heterogeneous class includes proteins with a

single or a partial MYB repeat, collectively assigned as „„MYB-related‟‟ that divided

into various subclasses (Rosinski and Atchley 1998). The single or partial MYB-repeat

or R3-type class includes TRIPTYCHON (TRY), CAPRICE (CPC) and MYBL2

likely to have evolved from R2R3-MYB genes (Fig 2.2 A1), are seen to be involved in


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control of cellular morphogenesis (Pesch and Hulskamp 2009) and secondary

metabolism control (Dubos et. al. 2008; 2010). The evolutionary older R1/2 type

includes the Circadian Clock Associated1 and Late Elongated Hypocotyl which

encode the core components of the central circadian oscillator (Lu et. al. 2011;

Hemmes et. al. 2012) (Fig. 2.2 A1).

A decade ago, Arabidopsis genome sequencing uncovered the comprehensive

description and classification of different transcription factors in plant world. It was

then the MYB transcription factor genes were explored and seriously targeted (Stracke

et. al. 2001). The MYB genes form one of the largest families, which have the most

numbers and functions in plants. The large size of this family in plants indicates their

significance in the control of plant specific processes. The increasing accessibility for

plant genome sequences has allowed comparisons and a better understanding of the

evolution of MYB transcription factor family (Dubos et. al. 2010; Du et. al. 2012;

2013). A number of MYB genes and their proteins have been examined in numerous

non-grass and grass family comprising approximately 304 members in Arabidopsis,

239 in grapevine (Vitis vinifera L.), 210 in poplar (Populus trichocarpa), 180

members in Brachypodium and 418 members in rice (Oryza sativa), maize (Zea

mays), wheat (Triticum astevum), Hordeum vulgare, Sorghum bicolour and sugarcane

using both genetic and molecular approaches respectively (Table 2.2). In the past

decade, the MYB genes have been extensively studied and have been found to be

involved in diverse physiological and biochemical processes including the regulation

of secondary metabolism (Borevitz et. al. 2000; Jin et. al. 2000; Nesi et. al. 2001),

control of cell morphogenesis and trichome development (Lee and Schiefelbein 1999;

Higginson et. al. 2003), regulation of meristem formation, floral and seed coat

development (Penfield et. al. 2001; Schmitz et. al 2002; Shin et. al. 2002; Steiner-

Lange et. al. 2003), and the control of the cell cycle (Ito et. al. 2001; Araki et. al.

2004). Some are also involved in various defense and stress responses (Vailleau et. al.

2002; Abe et. al. 2003; Denekamp and Smeekens 2003; Nagaoka and Takano 2003),

in light and hormone signaling pathways (Gocal et. al. 2001; Newman et. al. 2004,

Chen et. al. 2012). All the known MYB proteins, including those which are found in

Drosophila, frogs, chicken, mice, yeast, humans, angiosperms and gymnosperms


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share a definite structural feature, a highly conserved DNA-binding domain called the

MYB-domain (Lipsick, 1996; Martin and Paz-Ares 1997) and typically consists up to

four imperfect amino acid sequence repeats (R) of approximately 51-53 amino acids,

each forming three alpha helices structure similar to the motif of prokaryotic

transcriptional repressors and eukaryotic homeodomains (Braun and Grotewold 1999;

Du et.al. 20012; Kranz et. al. 2000; Stracke et. al. 2001). The second and third helices

are located at the C-terminus adopt a variation of the helix–turn–helix (HLH)

conformation with three regularly spaced tryptophan (W/hydrophobic) residues (Fig.

2.2). Furthermore, these regularly spaced tryptophan residues build a central

tryptophan cluster in the three-dimensional HTH fold

Table: 2.2 The MYB and MYB-related ESTs from non-grasses and grasses

collected from the Plant Transcription Factor Database, modified on August

2013

No. Plant species MYB genes MYB-related genes

NON-GRASSES

1 Arabidopsis lyrata 149 64

2 Arabidopsis thaliana 168 97

3 Gossypium hirsutum (Cotton) 76 61

4 Glycine max (Soybean) 369 265

5 Malus domestica (Apple) 238 146

6 Picea glauca (White spruce) 46 35

7 Pinus taeda (Pine) 35 21

8 Populus trichocarpa (Black cottonwood) 212 213

9 Ricinus communis (Castor) 62 98

10 Nicotiana tabacum (Tobacco) 48 38

11 Vitis vinifera (Grapes) 138 114

GRASSES

1 Brassica napus 65 70

2 Hordeum vulgare (Barley) 99 168


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No. Plant species MYB genes MYB-related genes

3 Oryza sativa subsp indica (Rice) 121 89

4 Oryza sativa subsp japonica (Rice) 130 106

5 Saccharum officinarum (Sugarcane) 36 38

6 Sorghum bicolor 132 116

7 Triticum astevum (Wheat) 103 105

8 Zea mays (Maize) 203 169

forming a hydrophobic core (Ogata et. al. 1994). The third helix of every repeat is the

“recognition helix” that makes direct contact with the DNA and intercalates in the

major groove so that other two helices bind specifically to the DNA sequence

recognition motif C/TAACG/TG (Ogata and Nishimura 1995; Ogata, 1998).

2.7 Why focus on MYB transcription factors

Numerous proteins with distantly related MYB domains do not appear to bind directly

to DNA, but rather act as components of multi-protein machines that modify different

histones. These MYBs participate in plant specific processes including primary and

secondary metabolism, developmental processes, control of cell fate determination

and regulation of the cell cycle and responses to different environmental stress

conditions (Table 2.3).

Several R2R3-MYB proteins are involved in regulation of different primary and

secondary metabolism. AtMYB11, 111 play an essential role in different flavonoid

biosynthesis (Stracke et. al. 2007); ZmMYBC1, AmMYB305, 340, AtMYB75, 113, 114

control anthocyanin biosynthesis in plant tissues (Paz-Ares et. al. 1987), AmMYB308,

330 are found to be regulating phenolic acid biosynthesis (Tamagnone et. al. 1998),

PsMYB26 from Pisum sativum is involved in the phenylpropanoid regulation (Jin and

Martin 1999) along with AtMYB12, NtMYBGR1 (Shinya et. al. 2007) and VvMYB5a

controls the tannins and lignins biosynthesis (Deluc et. al. 2006), whereas AtMYB52,

54, 69 are proposed to regulate the lignin, cellulose, xylan biosynthesis and are

positive regulators for cell wall thickening in fiber cells (Jin et. al. 2000). In Oryza

sativa, five MYB genes, OsMYB1-5, are seen to be transcribed during seed


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development (Suzuki et. al. 1997). Also the seed-specific AtTRANSPARENT TESTA2

(TT2) from Arabidopsis regulates proanthocyanidin accumulation in developing seeds

and ATR1 regulates the tryptophan biosynthesis in Arabidopsis (Celenza et. al. 2005).

The P. hybrida MYB gene, PhMYB3, function as dedicated transcriptional activators

in regulating the synthesis of different anthocyanin (flavonoid)-related compounds

(Solano et. al. 1995).

The role of R2R3-MYB proteins in developmental processes is also widely

characterized in different plant species. AtMYB37, 38, 84 are dedicated regulators of

axillary meristem formation (Keller et. al. 2006), AtMYBGL1, AtMYB0 and AtMYB23

positively regulate trichome development in shoots (Kirik et. al. 2005), whereas

AtMYB91 and CotMYBA in G. hirsutum; regulates the shoot morphogenesis and leaf

patterning (Byrne et. al. 2000), AmMYBMIXTA and AtMYB16 are positive regulators

of conical cell development in A. majus and Arabidopsis respectively (Baumann et.

al. 2007; Jaffé et. al. 2007), whereas AmMYBPHAN functions in dorsoventral

determination and growth in plant (Jin and Martin 1999). Many MYB TFs from

tobacco, cotton and soybean display gene-specific expression patterns in tissues and

flower buds (Shin et. al. 2002; Feller et. al. 2011); stem elongation, anther

development and seed development (Woodger et. al. 2003); anther tapetum, stigma

papillae, and lateral root primordial (Preston et. al. 2004). Moreover, R2R3-MYB

genes are also reported to be involved in the signal transduction pathways of abscisic

acid (Abe et. al. 2003) which play a significant role in the adaptation of vegetative

tissues to abiotic environmental stresses such as drought and high salinity, salicylic

acid (Raffaele et. al. 2008), gibberellic acid (Gocal et. al. 2001; Murray et. al. 2003),

and jasmonic acid (Lee and Schiefelbein 2001) as well. Further the rice OsMYB4 gene

is seen to adapt cold conditions whose constitutive expression in Arabidopsis resulted

in improved cold and freezing tolerance (Vannin et. al. 2004). Plants have developed

various strategies to respond to unfavourable environmental conditions. Variety of

active defence mechanisms are triggered on to protect themselves from any such

condition. Functional analysis of plant R2R3-MYB TFs indicate that they regulate

numerous processes, including responses to environmental stress. Drought stress is

one such condition and it affects almost all plant functions including growth and


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Table: 2.3 Some of the MYB transcription factors with their multifunctions

Plant Gene Function Author

Sugarcane PScMYBAS1 response to dehydration, salt,

cold, wounding

Prabu and Prasad

2012

Arabidopsis

AtMYB11, 111 flavonoid biosynthesis Stracke et. al. 2007

AtMYB75, 113,

114

anthocyanin biosynthesis Paz-Ares et. al. 1987

AtMYB12 phenylpropanoid regulation Shinya et. al. 2007

AtMYB52, 54, 69 Lignin and cellulose regulation Jin et. al. 2000

AtMYB37, 38, 84 axillary meristem formation Keller et. al. 2006

AtMYBGL1,

AtMYB30 and 23

regulate trichome development Kirik et. al. 2005

AtMYB91 regulates shoot morphogenesis Byrne et. al. 2000

AtMYB16 conical cell development Jaffé et. al. 2007

AtMYB2 Drought resistance Abe et. al. 2003

AtMYB68 Temperature resistance Feng et. al. 2004

Rice

OsMYB4 adapt cold conditions Vannin et. al. 2004

OsMYB2P-1 Pi-dependent regulator Dai et. al. 2012

OsMYB7 oxygen deficit response Mattana et. al. 2007

OsMYB2 tolerance to salt and dehydration Yang et. al. 2012

OsMYB1R response to all stresses Chen et. al. 2014

OsMYB48-1 drought and salinity tolerance Xiong et. al. 2014

Maize

ZmMYB-IF35 secondary metabolic pathways Heine et. al. 2007

ZmMYB regulation of flavonoid Grotewold, 2005

ZmMYB gene regulation Feller et. al. 2011

Sorghum SbMYBy1 chalcone synthase Boddu et. al. 2006


23

Plant Gene Function Author

Wheat

TaMYB33 drought stress Qin et. al. 2012

TaMYB1 expressed under hypoxia Yan et. al. 2009

TaMYB32 tolerance to salt stress Zhang et. al. 2012

TaMYB30-B PEG stress Zhang et. al. 2012

TaMYB1 cold, PEG and salt Lee et. al. 2007

TaMYB3R1 tolerance under PEG condition Cai et. al. 2011

TaMYB32 drought stress Zhang et. al. 2012

Others

BcMYB1 drought stress Chen et. al. 2005

BrMyb pathogen infection and plant

development

Yu et. al. 2012

PoMYB134, 182 flavonoid metabolism Yoshida et. al. 2015

CmMYB2 drought tolerance Shan et. al. 2012

MtMYB, MtMYB14 proanthocyanidin accumulation Liu et. al. 2014

CotMYBA regulates shoot morphogenesis Byrne et. al. 2000

TaMYB14, biosynthesis of

proanthocyanidins

Hancock et. al. 2012

GbMYB5 drought tolerance Chen et. al. 2015

PpMYB10.4 anthocyain accumulation Zhou et. al. 2014

AmMYB305 floral organ development Liu, 2010

Ta: Trifolium arvense ; Cot: G. hirsutum; Mt: M. truncatula; Cm: Chrysanthemum;

Po: Populus; Br: Bracissca rapa; Bc: Boea crassifolia; Gb: Gossypium barbadense,

Am: Antirrhinum majus.

development (Ambawat et. al. 2013). For instance, AtMYB2 is transiently induced by

dehydration and functions as transcriptional activator in ABA (abscisic acid)

inducible gene expression during drought (Abe et. al. 2003); AtMYB68 is modulated

by temperature (Feng et. al. 2004); BcMYB1, from Boea crassifolia, was also strongly

induced by drought stress (Chen et. al. 2005). Also the tobacco MYB1 gene is seen to

encode a signalling component downstream of salicylic acid that participate in

transcriptional activation of pathogenesis-related genes and plant disease resistance

(Yang and Klessig 1996). Bracissca rapa plant TF, BrMyb, is shown to play a role not


24

only in plant development but also in the response to pathogen infection and other

stresses (Yu et. al. 2012). The AtMYB2 gene in association with probe like salt-

inducible CaM isoform regulates the expression of salt- and dehydration-responsive

genes in Arabidopsis (Yoo et. al. 2005). Also the combinational regulations have

successfully been used to predict new MYB/BHLH interactions for flavonoid

biosynthetic pathway in A. thaliana proteins which is conserved amongst higher

plants. Lately, R2R3-MYB transcription factor, VvMYBPAR, from grapes has shown

enhanced proanthocyanidin biosynthesis with consitutuve expression in Arabidopsis

(Koyama et. al. 2014), the R2R3-MYB transcription factors MYB134, MYB182 from

Populus are seen to play an important role in the flavonoid metabolism and down

regulates the production of proanthocyanidin in transgenic Arabidopsis (Yoshida et.

al. 2015) and the overexpression of MtMYB5 and MtMYB14 of M. truncatula in

Arabidopsis has strongly induced proanthocyanidin accumulation (Liu et. al. 2014).

Moreover, expression of the Chrysanthemum R2R3-MYB transcription factor

CmMYB2 has showed enhanced drought tolerance revealing increases hypersensitivity

towards the ABA in transgenic Arabidopsis (Shan et. al. 2012), Brassica Oleracea

MYB transcription factor has seen to be induced by lower temperatures and accelerate

biosynthesis of anthocyanin (Zhang et. al. 2012) whereas the TaMYB33 from Triticum

aestivum has seen to over-expression during the drought stress condition in transgenic

Arabidopsis (Qin et. al. 2012). Thus, the coming years are apparent to onlook a

growing understanding of the conserved MYB transcription factors which may

participate at comparable gene regulatory networks across plant species under any

stress conditions.

2.8 Role of MYB TFs in grasses under stress conditions

The large and ubiquitous Poaceae family (syn: Gramineae/ true grasses) comprise of

monocotyledonous flowering plants stretched over wetlands, forests and tundra

habitats. The family is estimated to compose around 20 % of the vegetation cover of

the earth. The physiological groups of the grasses follow two (C3 and C4)

photosynthetic pathways for carbon fixation. The C4 grasses have a photosynthetic

pathway linked to specialized Kranz leaf anatomy that particularly adapts them to hot


25

climates and atmospheres low in carbon dioxide. The economically important

members of the family include the grain crops like rice, maize, wheat, barley,

sorghum and leaf and stem crop like sugarcane, bamboo etc (http://en.wikipedia.org).

Due to exposure of these members to different and altering climatic conditions the

crop yield is tremendously affected. The loss of crop yield is mostly due to drought,

salt and temperature conditions, disease attack by pathogens, insects, nematodes,

viruses and bacteria. Farmers all over the world are facing problems to find remedies

for getting rid of this loss. Thus advanced molecular approaches were pioneered by

the researches for the betterment of the crop yield and aid for farmers. Numerous

groups of scientists then focused on the transcriptional regulation of these crops for

understanding the pathway taken up for tolerating these stress conditions. Many TFs,

including DREB1/CBF, DREB2, AREB/ABF, NAC, MYB, and WRKY can be used to

improve stress tolerance to abiotic and biotic stresses in grasses. However, couple of

challenges have to be considered while working with TFs for genetic engineering of

stress tolerance. Firstly, an efficient expression system, including reliable promoters,

may be required for each group as constitutive promoters may not function or have

negative effects on plant growth and development (Prabu and Prasad 2012). Secondly,

the prime need to establish promising systems to appraise the stress tolerance in

transgenic grasses under field conditions. Some of the worked out projects applied

cDNA suppression subtraction hybridization (cDNA-SSH), cDNA-AFLP, cDNA-

microarray techniques in response to biotic and abiotic stress conditions in various

plant systems. To comprehend the molecular basis of specific plant pathogen

interactions, genes responding to the pathogen attack (Ustilago

scitaminea or Bipolaris sacchari ) in sugarcane were identified by using cDNA-AFLP

technique (Borras-Hidalgo et. al. 2005). Totally 62 differentially regulated genes were

identified, of which 10 were down-regulated and 52 were induced which further

aimed at understanding sugarcane pathogen defense; whereas the deciphered 83

SCGS-specific gene fragments by genomic-SSH towards its molecular basis of

virulence of phytoplasma in sugarcane. Furthermore, the transcription factor gene

(SoMYB18, Accession No: FJ560976) identified and was characterised from the

phytoplasma infected sugarcane SSH library (Kawar et. al. 2010).


26

Many studies have been reported on effects of abiotic stress like salt and drought in

sugarcane. A study reported use of cDNA-RAPD approach to generate the transcripts

of sugarcane plantlets exposed to 2 % NaCl (Pagariya et. al. 2010). Around 335

differentially expressed transcript-derived fragments were monitored by ribotyping of

which 156 were up- and 85 down-regulated and functionally categorized under

metabolism, DNA and RNA processes, signal transduction or cell rescue or defense,

transcriptional regulation and hypothetical proteins. Similar studies carried out in

sugarcane have revealed and identified many stress responsive gene expression in

response to salt and PEG conditions (Patade, 2009; Patade et. al. 2011). The NHX

(sodium proton antiporter), SUT1 (sucrose transporter1), PDH (proline dhydrogenase)

and CAT2 (catalase2) were observed in response to 2-4 h PEG stress and shaggy-like

protein kinase were identified during the salt stress condition. The PCR-based cDNA

SSH studies reported 158 differentially expressed genes under water deficit stress

which encoded proteins for cellular organization, protein metabolism, signal

transduction, and transcription (Prabu et. al. 2011a). Further, the cDNA library

screening gave the sugarcane stress-related MYB transcription factor gene and

promoter, ScMYBAS1 and PScMYBAS1, respectively (Prabu and Prasad 2012). The

recent protein docking studies were put forth to elucidate the sequence-to-structure-to-

function paradigm of ScMYBAS1 by understanding the putative three-dimensional

structure using threading assembly refinement (I-TASSER) server, PROCHECK,

Verify-3D, PROMOTIF and ProSA programs (Prabu and Prasad 2011b). Also the

stress related MYB promoter (PScMYBAS1, 1,033 bp) isolated was characterized by a

series of deletion derivatives from the transcription start site (-56, -152, -303, -442, -

613, -777, -843, -1,033) and fused to the uidA reporter gene (GUS) to analyze by

Agrobacterium-mediated transient transformation in tobacco leaves. The

results projected about ~twofold to ~fourfold increased induction of GUS in response

to dehydration, salt, cold, wounding response and hormone (SA, MeJA) treatments.

Thus the regulation of ScMYBAS1 under PScMYBAS1 promoter is seen to provide a

new stress-inducible system in transgenic plants.

The identification of host genes involved in defense responses are one of the most

critical steps leading to the elucidation of disease resistance mechanism in plants. The


27

tropical japonica rice cultivar (Oryza sativa cv. Drew) infected with blast fungus

(Pyricularia grisea) was subjected to cDNA library preparation isolating 22 genes

expressed in response to the pathogen (Xiong et. al. 2002). Further, the database

searches of these genes encoded the putative functions for zinc finger proteins, ABC

transporters and other potential defense-signaling components. Another study carried

out by Shim et.al. 2004 using cDNA-SSH to identify fungus (Magnaporthe grisea)

stress induced genes in Oryza minuta revealed 377 cDNA clones which were

classified into seven different groups using microarray data-derived expression

patterns. High hydrostatic pressure (HHP) is considered an extreme thermal–physical

stress which affects the multiple cellular activities of plant. To understand the

molecular mechanism of plant response to HHP, the constructed forward and reverse

subtracted cDNA-SSH libraries of rice seeds treated with 75 MPa hydrostatic pressure

for 12 h (Liu et. al. 2008). Of 45 unique isolated genes, 29 clones were significantly

similar to known genes with functions involved in metabolism, defense response,

transcriptional regulation, transportation regulation, and signal transduction. Being the

first of its kind the expression profile of this gene would provide useful information

regarding molecular processes, including alteration of metabolism and adaptation

response caused by HHP. The inevitable research in understanding the mechanisms

underlying stress responses and new stress-tolerance genes in rice (Oryza sativa L.)

have helped to analyse globally the genome expression profile of indica cv.Pei‟ai64S

subjected to cold, drought and heat stresses (Chen et. al. 2012). Expression profiles

obtained from leaf and panicle generated 51,279 transcripts, of

which OsMYB1R, was highly induced in response to all stresses. Sequence analysis

showed that the cDNA encoded a protein of 489 amino acid residues with molecular

weight (M.W.) ≈ 56 kD and pI ≈ 6. Bioinformatics analysis revealed the protein

having a conservative domain of the MYB gene family. Thus, concluding that

OsMYB1R was novel candidate gene which was seen to be involved in stress

tolerance in rice. Interesting findings made by projects exposure of rice seedlings to

phosphate (Pi)-deficient medium (Chen et. al. 2014). The overexpression of

OsMYB2P-1 in Arabidopsis (Arabidopsis thaliana) and rice was seen to enhance the

tolerance to Pi starvation, while suppression of OsMYB2P-1 by RNA interference in


28

rice rendered sensitivity to Pi deficiency in transgenic rice. The OsMYB2P-1 gene

may seem to act as a Pi-dependent regulator in controlling the expression of Pi

transporters (Dai et. al. 2012). These interesting findings demonstrated OsMYB2P-1 to

be a novel R2R3-MYB transcriptional factor associated with Pi starvation signalling in

rice. Furthermore, an interesting study in rice with R2R3 MYB, OsMYB7, illustrated a

diverse role encoding two different TFs as a result of regulated splicing in response to

oxygen deficit response (Mattana et. al. 2007). This spliced OsMYB7 mRNA is seen

to encode MYB factor showing canonical features, while the unspliced mRNA

encoded a transcription factor (MYBleu) composed of an incomplete MYB domain

followed by a short leucine zipper. (Caruso et. al. 2012). The R2R3-MYB, OsMYB2

enhanced the tolerance to salt and dehydration stress conditions and the transgenics

showed sensitivity to ABA response in rice (Yang et. al. 2012).

In wheat, SSH and high density membrane techniques were employed to analyse

genes induced by water stress. The research carried out comprehensively was to

understand the

genetic bases of drought resistance and find the key genes related to drought

resistance in wheat (Wang et. al. 2007; 2010). Total of 181 positive clones were

obtained, of which 17 differentially expressed genes were highly homologous with

those induced by abiotic or biotic stresses in plant. The flavonoid biosynthesis

pathway products like polyphenol compounds, phlobaphene orproanthocyanidin were

reported to be part red pigments in wheat grains. Further the expression of these red

pigment genes (R-genes) were seen to be located on the long arms

of chromosomes 3A, 3B and 3D and were found to be the transcriptional activators of

the flavonoid synthesis genes (Himi et. al. 2005). The identification of the MYB-type

transcription factors, TaMYB10-A1 on 3A, Tamyb10-B1 on 3B and Tamyb10-D1 on

3D chromosomes, in the same region of R loci, were seen to be expressed

predominantly in the developing grains. Oxygen deficiency being a major stress in

waterlogging plants, the comprehensive study was of the low-oxygen-signalling

pathway for adaptation and survival (Lee et. al. 2007). The genes related to the

oxygen concentration in roots environment were investigated by transcriptional

expression in vitro. The TaMYB1 gene was expressed under hypoxia and continued

http://europepmc.org/abstract/AGR/IND43765372/?whatizit_url_go_term=http://www.ebi.ac.uk/ego/GTerm?id=GO:0005694

http://europepmc.org/abstract/AGR/IND43765372/?whatizit_url_Chemicals=http://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI%3A47916


29

until approximate anoxia. The TaMYB1 transcriptional levels in roots increased

gradually and strongly relate to the oxygen concentration in root environment and its

response to abiotic stresses. The cDNA library constructed with SSH approach using

the wheat near isogenic line TcLr19 inoculated with Puccinia recondita f. sp. tritici.

The 1337 positive ESTs were screened using reverse Northern blotting and 237

selected clones were subjected to similarity analysis for comparing with BLAST

results (Yan et. al. 2009). Most of the clones were related to biological processes

including signal transduction, transcriptional regulation and hypersensitive responses.

Heat responsive genes from wheat under high temperature stress were elucidated

(Chauhan et. al. 2010) through construction of subtractive cDNA libraries. Total of

5,500 ESTs were generated and BLAST analysis showed homology of 10 genes with

a new heat shock protein factor and putative signalling molecules. Additionally, the

first comprehensive study was reported for the MYB gene family in Triticeae, sixty

full-length cDNA sequences were isolated from wheat and categorized under

R1R2R3-MYB, R2R3-MYB and MYB related members. The expression analysis of the

genes during abiotic stress identified an overexpression of a salt-inducible gene,

TaMYB32, and enhances during the tolerance to salt stress in transgenic Arabidopsis

(Zhang et. al. 2012). Reports in Sorghum suggest the genomic sequence

characterization of yellow seed1 with two duplicate genes (y1 and y2) separated by

9.084 kbp intergenic region (Boddu et. al. 2006). The y1 gene was seen to encode

R2R3 type of MYB domain protein which regulated the expression of chalcone

synthase, isomerase and dihydroflavonol reductase genes which were a part of the

biosynthesis of 3-deoxyflavonoids. Osmotically stressed maize seedlings were studied

by constructing the full-length cDNA library using modified CAP trapper method (Jia

et. al. 2006). The generated 2073 full-length cDNAs which were further analyzed by

sequencing. Expression analysis of these 2073 full-length maize cDNAs was carried

out by cDNA macroarray and illustrated 79 genes to be upregulated and 329 down

regulated by stress treatments. Of these 30 genes contained ABRE, DRE, MYB, MYC

abiotic-responsive cis-acting elements in their promoters concluding the role of

transcription factors in plant responses to drought stresses. The characterization of

ZmMYB-IF35 isolated from maize was reported (Heine et. al. 2007) to bind and


30

activate transcription in yeast providing interesting findings regarding transcriptional

activation and novel insights on regulation of secondary metabolic pathways.

Moreover, studied related to salt sensitive maize inbred lines, NC286 and Huangzao4,

treated with 200mM NaCl revealed early responsive genes by SSH approach (Ding et.

al. 2009). Total of 141 unique genes were significantly profiled and similarity analysis

showed involvement in broad spectrum of biochemical, cellular, and physiological

processes. They were assigned to 14 categories based on their biological functions.

The transcriptional activity of the R2R3-MYB factors is dependent in vivo on protein-

protein interactions. Advances in studies of MYB TFs have helped to recognize the

combinational regulations between genetic and direct physical interactions suggesting

an intimate functional relationship between MYB proteins and bHLH proteins. The

regulation of flavonoid biosynthetic gene expression in Zea mays provides one of the

best-described examples of combinatorial gene regulation. (Grotewold, 2005; Feller

et. al. 2011).

2.9 Sugarcane MYB transcription factors

Different molecular approaches are being applied to segregate the specific trait genes,

for example stress tolerant, sucrose accumulation genes, transcription factors,

promoters; and check their efficacy with regards to its regulation and expression. In

our early attempts, suppression subtractive hybridization (SSH) approach was put

forth to isolate up regulated responsive cDNAs (ESTs) in sugarcane during water-

deficit stress (Prabu et. al. 2011a) studies to elucidate the transcriptional regulatory

mechanisms. Functional characterization of these genes categorize the ESTs under

enzymes, carbohydrate metabolism, and cell signaling, ion transportation and

transcription factors using the NCBI database. One ScMYBAS1-3 gene (accession no.

EU670236) was isolated in response to water deficit stress and its in silico

illustrations were projected (Prabu and Prasad 2011b) and one SoMYB18 gene

(accession no FJ560976) in response to phytoplasmal infection (Kawar et. al. 2010).

Further more efforts were made to explore the molecular adaptation mechanisms of

stresses and to strengthen stress tolerance in this plant (Watt, 2003; Patade, 2009;

Gupta et. al. 2010; Kawar et. al. 2010; Pagariya et. al. 2010, Prabu et. al. 2011a;


31

Sundararaman et. al. 2014). The sugarcane MYBs (ScMYBs) isolated provided a vast

information regarding their Gene Ontology, expression in salt and drought stress with

a wide bioinformatical analysis revealing the better R2 and R3 domains and

evolutionary phylogenetic relation ship with other database homologs (Sundararaman

et. al. 2014). The information provided assisted in considering these MYB

transcription factor family from sugarcane multifunctional and more promising. The

identification of genes and promoters with suitable abiotic and biotic stress

inducibility will essentially aid to drive stress-inducible gene expression in sugarcane.

One such study reported included the functional characterization of sugarcane MYB

stress inducible promoter, PScMYBAS1, beneficial for the plant transgenic system

(Prabu and Prasad 2012).

As sugarcane is time again attacked by different bacterial, fungal and viral diseases

and environmental stress conditions the cost of productivity of the crop has been in

question. It was experimented to hybridize the genus Saccharum with closely related

species but the gene flow in the hybrids emerged to be very stumpy. To enhance the

insight of the response to the changing environments and the role of MYB genes in the

control of plant-specific processes, isolation and analysis of MYB genes from the wild

relative species of sugarcane has now been an official research.

2.10 Plant Transformation

Plant transformation technology has become a core research area of plant biology and

a practical tool for the crop cultivar improvement. Various methods for stable

introduction of the novel genes into the nuclear genomes of over 120 diverse plant

species have been practiced (Birch, 1997). Due to considerable developments in this

technology large number of transgeneic crops have been released for commercial

production within last two decades. Choice of plant transformation vectors and

methodologies have significant improvement to increase the efficiency of the plant

transformation and to achieve stable expression of transgenes in plants (Pamela,

1993).

Recent advancements in plant genetic engineering have made gene transfer achievable

into crop plants from unrelated plants and also from the nonplant organisms.


32

Consequently, many crop species are being genetically modified for betterment of the

agronomical traits, including biotic and abiotic stress conditions, better nutritional

values, and other desirable qualities.

2.10.1 Agrobacterium-mediated plant transformation

Agrobacteria are the prominent and beneficial soil-borne bacterial plant pathogens.

The A. tumefaciens has the property to naturally infect the wounded sites in the

dicotyledonous plant which consequently form the crown gall tumors was reported

way back in the early nineties (Smith and Townsend 1907). The transfer of the

foreign gene in plants allowing genetic manipulation is the trait of Agrobacterium.

This bacterium has the exceptional ability to transfer the harboring DNA segment (T-

DNA), the tumor-inducing (Ti) plasmid, into the nucleus of infected plant cells

(Nester et. al. 1984). This T-DNA is then stably integrated into the host genome and

transcribed thus causing the crown gall disease (Binns and Thomashaw 1988). The

transfer of the T-DNA takes place with help of the two oncogenic genes enzyme

products which synthesize auxins and cytokinins for the tumor formation. These

compounds were observed to be synthesized after the condensation between the

amino acids and sugars and oozed by the crown gall cells and A. tumefaciens consume

it as carbon and nitrogen sources for growth (Hooykaas and Schilperoort 1992). The

genes for the opine catabolism present outside the T-DNA assist the transfer from the

bacterium to the plant cell by plasmid conjugative method. (Zupan and Zambrysky

1995; Gelvin, 2003). Research has been carried out in sugarcane crop under various

aspects to confer tolerance, may it be abiotic or biotic stress. This approach of

transformation of resistance genes into sugarcane crop may have positive impact on

the sugarcane yields. The evidence of generation of the first transgenic sugarcane

lines resistant to stem-borer attack was reported (Arencibia et. al. 1997). Even many

other monocotyledonous plants majorly the economic crops, vegetables, ornamental,

medicinal, fruit, tree and pasture plants (Birch, 1997) have been enhanced using

Agrobacterium-mediated or direct transformation methods for various aspects.

Recently a consistent and proficient vacuum infiltration-assisted method consisiting

Agrobacterium-mediated genetic transformation (VIAAT) protocol was developed for


33

soybean cultivars which produced transgenic soybean plants through somatic

embryogenesis (Thankaraj et. al. 2013). Recently the transformation efficiency

observation using the Agrobacterium- mediated transformation was reported to be 95

% in transgenic tobacco plants (Pathi et. al. 2013). Also the transformation efficiency

of five Agrobacterium tumefaciens strains, GV2260, LBA4404, AGL1, EHA105,

C58C1, harboring the plasmid pBin19 containing beta-glucuronidase uidA gene under

35S CaMV promoter in Nicotiana tabaccum (Bakhsh et. al. 2014).

Improved genetic transformation protocol was introduced for the recalcitrant indica

rice cultivar IR64 using Agrobacterium strain LBA4404 mediated genetic

transformation () which provided successful tannsgenic plants (Sahoo and Tuteja

2012). Gene Am-SOD isolated from Avicenia mariana, a mangrove species was agro

mediated-transformed to examine the excess salt tolerance developed in the transgenic

rice plants (Sarangi et. al. 2011). Various cultivars of wheat were targeted to break the

recalcitrant nature in vitro. Agrobacterium mediated- transformation using strains

A281, GV3101, ABI, EHA101, EHA105, AGL0, M-21 were performed to generate

transgenic plants for crop betterment (Pérez-Piñeiro et. al. 2012). Wheat immature

embryos in a liquid culture of Agrobacterium tumefaciens were promoted to develop

showing successful transgenic plants (Sparks et. al. 2014). Soybean being a model

legume crop recent introduction of agro-mediated and particle bombardment

transformation procedures are being practiced. The agro-mediated transformation

showed better efficiency and simple method to practice is commonly used to generate

transgenic crops (Lee et. al. 2012). Recent studies provide information for twenty

soybean genotypes originated from different regions in China for transient infection,

regeneration capacity, and stable transgenic efficiency using the agro-mediated

transformation procedure. The three genotypes showed ~4.5 % efficient

transformation using Agrobacterium strain EHA101 (Song et. al. 2013). Another

Agrobacterium mediated transformation protocol for tomato (Solanum lycopersicum

L.) using the DREB1A gene under the Rd29A promoter in the pCAMBIA2301 binary

vector was performed to optimize various parameters like type and concentrations of

the hormones. The protocol used could rapidly generate effiecient transgenic tomato

plants (Manmohan et. al. 2011). Current studies have reported that the agro-mediated


34

transformation protocol was used to transform ySAMdc gene under the E8 fruit

promoter to generate the putative tomato transgenics which showed genetically

enhanced lycopene content (Somayaji et. al. 2014). Lately, a robust agro-mediated

transient expression system, named the AGROBEST for seedling transformation

showed simple, fast, reliable system which could assist to dissect the molecular

mechanisms involved in Agrobacterium-mediated DNA transfer (Wu et. al. 2014).

2.10.2 Tobacco model plant for MYB transcription factor transformation

experiments

Tobacco (Nicotiana tabacum L.) 2n = 4X = 48 is a natural allotetraploid which is the

product of two diploid 2n = 24 progenitors, Nicotiana tomentosiformis and Nicotiana

sylvestris (Hajdukiewicz et. al. 1994). Having short generation period, the plant

attains maturity within 3 months with millions of seeds per plant. The plant was used

for various experiments for transferring foreign DNA by vector mediated transfer and

were the first transgenic plants ever to be produced . Thus, the experimentations

related to the gene stability, gene transformation and expression were routinely

carried out in tobacco (Suzuki and Takebe 1976). Many transformations were carried

out in tobacco using Agrobacterium system for various traits. Tobacco chloroplasts

and the Agrobacterium

mediate transformation combination was used to introduce into the higher plants by

site

specific homologous recombination (Suzuki and Takebe 1976 and Ganapathi et. al.

2004).

Lately, a very simple highly efficient and fast protocol was put forth for regeneration

method for the introduction of any foreign gene directly in tobacco through direct

somatic embryogenesis (Pathi et. al. 2013). Various expression studies have been

reported such as R2R3-MYB transcription factor, TaMYB14, from Trifolium arvense

provided confirmation that this transcription factor is related with the regulation of

biosynthesis of proanthocyanidins in the legumes. Transformation of the TaMYB14 in

Nicotiana tabacum showed constitutive expression, synthesis and accumulation of

proanthocyanidins in leaves (Hancock et. al. 2012).


35

Furthermore, Arabidopsis MYB transcription factor, AtMYB111 results in the

overexpression in transgenic tobacco and enhanced the expression of genes

responsible for the phenylpropanoid pathway which may lead to the elevated content

of flavonols in presence of light as in constrast to the wild type plants (Pandey et. al.

2014), a R2R3-type MYB transcription factor gene in Gossypium barbadense,

GbMYB5, conferred drought tolerance in the transgenic tobacco by enhanced

accumulation of proline and antioxidant enzymes (Chen et. al. 2015). The EsMYBA1

transcription factor from a traditional Chinese medicine Herba epimedii (Epimedium),

was transformed in tobacco so as ensure the expression of this TF in the pathway of

flavonoid and essentially overexpressed anthocyanin accumulation in the flavonoid

biosynthesis and regulation in transgenic tobacco (Huang et. al. 2013). An MYB

transcription regulator from Prunus persica PpMYB10.4 was identified to direct the

anthocyanin pigmentation in the peach leaf. The transient overexpression of

PpMYB10.4 in the tobacco and in peach leaf led to induce anthocyain accumulation

(Zhou et. al. 2014). The MYB transcription factor from the grey mangrove AmMYB1

should constitutive expression in the transgenic tobacco plants for better tolerance to

NaCl stress (Ganesan et. al. 2012). The MYB from Antirrhinum majus, AmMYB305,

showed transient expression in tobacco and play important role in floral organ

development and its maturation (Liu, 2010).

2.11 Semi-quantitaitve and tissue-specific expression studies

Expression studies prove the understanding of any gene under various conditions such

as, stress, organ development, biosynthetic pathways for various flavonoid and

anathocyanin synthesis etc. The MYB transcription factor family has been a

multifunctional in regards with the expression under various circumstances (Dubos et.

al. 2010).

To comprehend the role of these transcriptions factors under these conditions

expression studies have been undertaken. In Arabidopsis, the MYB transcription

factor, AtMYB111, has shown to express in tobacco to enhance flavonoid

biosynthesis. The AtMYB111 transgene was confirmed by PCR amplification in the

transgenic tobacco lines and its expression was performed through semiquantitative


36

RT-PCR for control and transgeneic lines thus confirming the anthocyanin content in

flower petals to be reduced in control as in comparison to transgenic tobacco plants

(Pandey et. al. 2014). Also the AtMYB12 has shown several fold higher expression in

transcript levels analysed by quantitative RT-PCR in transgeneic tobacco and in the

tomato lines compared to the control plants (Luo et. al. 2008). The EsMYBA1 was

preferentially seen to be expressed in red leaves which contain higher content of

anthocyanin. (Huang et. al. 2013). The expression of OsMYB48-1 was seen to express

in various tissues, including root, stem, sheath, leaf, panicle and in roots at both

seedling and reproductive stages. Moreover, OsMYB48-1 was also seen to be induced

by multiple abiotic stresses such as cold, dehydration, H2O2, PEG, ABA and NaCl,

thus revealing positive role in drought and salinity tolerance by regulating the stress-

induced ABA synthesis (Xiong et. al. 2014). Tissue-specific expression pattern

analyses of 36 R2R3-type MYB genes from wheat confirmed the predicted orthologs

to be similar to Arabidopsis genes under stress conditions (Cai et. al. 2012). To

comprehend the expression levels of TaMYB30-B under PEG stress conditions, the

transgenic Arabidopsis lines were characterized for the overexpression of TaMYB30-

B and revealed to show tolerance towards drought stress during the germination and

seedling stages (Zhang et. al. 2012). A potential MYB gene (TaMYBsdu1) showed

significant up-regulation in leaves and roots of wheat plants when subjected to long-

term drought stress. Additionally, this MYB showed elevated transcript abundance in

the salt-tolerant genotype than in control under salt stress thus, posing TaMYBsdu1 as

regulator for wheat adaptation to both salt and drought stresses. Also TaMYB1

showed resistance to abiotic stresses like cold, PEG and salt (Lee et. al. 2007),

TaMYB3R1 showed higher tolerance under PEG condition (Cai et. al. 2011),

TaMYB32 showed tolerance towards drought stress (Zhang et. al. 2012).

2.12 Proteomics

Proteins are vital parts of living organisms, as they are the main components of the

physiological metabolic pathways of cells. The term proteomics was first coined in

1997 to make an analogy with genomics, the study of the genome (James, 1997).

Proteomics is one of the large-scale study of proteins, particularly their structures and


37

functions (Anderson and Anderson 1998; Blackstock and Weir 1999). Proteomic

analysis offers an added approach to potentially increase the understanding of the

molecular mechanisms of any plant subjected to any stress conditions. Proteomics is a

powerful, advanced and imminent technique applied in the sugarcane crop research.

The technique follows a basic principle to segregate complex protein mixtures to

produce visible results. Changes in protein profiles in response to different

environmental effects and diseases can be resolved with this method (Dhingraa et. al.

2005). Efforts are being set forth to analyze genes discovered from various methods

from sugarcane leaves under salt and water-deficit stress, pathogen, fungal and

bacterial infection. This technique is highly in demand as the gene function and its

physiological behavior can be analyzed effectively.

2.12.1 Approaches of Proteomics under stress conditions

For understanding or determining the function of any gene, identification of the

proteins encoded is very essential (Abbasi and Komatsu 2004). Investigating the

outcome of the stress induction in crop is important and can be studied more

efficiently at the protein level. Many proteomic approaches offer powerful tools

which have been implemented for systemic study of proteins isolated under stress

conditions in plants (Agrawal and Rakwal 2008; Cho et. al. 2010). The most popular

strategies used in proteomics is the two-dimensional electrophoresis and matrix-

assisted laser desorption/ionization-time of flight mass spectrometry which concludes

the differential and comparative expression of proteins expressed in plants (Komatsu

and Yano 2006; Agrawal et. al. 2009; Ding et. al. 2011; Kim et. al. 2011; Falvo et. al.

2012). pointed out that the one dimensional approach is seen to present a unique

prospect to clone and exemplify a large amount of proteins expressed by the

sugarcane genome. (Ramagopal, 1989). The 1-DE with immobilized pH gradients

facilitates the separation of intricate mixture of proteins according to isoelectric point,

molecular mass and relative abundance (Gorg et. al. 2004). Several current proteomic

studies have been executed on various crops under different abiotic stresses, such as

osmotic stress in rice and rapeseed (Zang and Komatsu 2007; Toorchi et. al. 2009),

drought in rice, wheat, sugar beet (Salekdeh et al. 2002; Vincent et al. 2005; Ali and


38

Komatsu 2006; Jorge et. al. 2006; Hajheidari et. al. 2007; Caruso et. al. 2009; Xu et.

al. 2009), high salinity in rice, (Parker et. al. 2006; Chen et. al. 2009; Jellouli et. al.

2010), low temperature in arabidopsis and winter rye (Amme et. al. 2006; Gao et. al.

2009). Sugarcane proteomic analyses have examined general protein polymorphisms

(Ramagopal, 1989), changes in protein expression after dedifferentiation of leaf tissue

in callus culture (Ramagopal, 1994) and drought-stress responsive proteins (Sugihario

et. al. 2002; Jangpromma et. al. 2010). Nevertheless, application of proteomics

knowledge in the studies of sugarcane needs to be expanded and meticulously

explored (Sugihario et al. 2002; Amalraj et. al. 2010; Jangpromma et. al. 2010). Many

researches are investigating abiotic stress related proteins isolated from sugarcane

using these proteomic tools in the

comparative based studies.

The protein expression system was used in sugarcane mainly to study protein

variation during leaf de-differentiation (Ramagopal, 1994) and to identify a drought-

inducible protein localized in the bundle sheath cells (Sugihario et. al. 2002). Studies

carried out in sugarcane revealed many proteins to be upregulated subjected to water-

deficit stress. These differentially expressed proteins were identified and studied the

identification of proteins from sugarcane under osmotic stress and obtained drought

inducible 22 kDa protein and ribulosebisphosphate carboxylase small chain proteins

(Rubisco small subunit) which were upregulated whilst ATP synthase delta chain and

isoflavonereductase-like proteins were down-regulated (Zhou et. al. 2011). Thus the

drought inducible proteins and energy metabolism and antioxidant defense-related

proteins play a vital role under osmotic stress conditions in sugarcane. Further, the

properties of these proteins were identified by using different bioinformatic tools and

analyses suggesting the proteins were more of acidic, unstable nature, transmembrane

and enriched with hydrophobic amino acids like leucine and alanine. Whereas, the

drought inducible 22-kDa protein was hydrophilic andnon-transmembrane protein

enriched with glutamic acid having involvement in adaptation to drought stress via

differentsignaling cascades. The obtained Peptide Mass Fingerprints (PMF) needs to

be compared with those present in the SWISS-PROT, NCBI and MSDB databases

using the MASCOT program (http://www.matrixscience.com). Studied carried out


39

sugarcane plants from three drought tolerant (RB92579, RB867515 and SP79-1011)

and three drought sensitive varieties (RB72454, RB855536 e RB855113) evaluated

the proteome plantlets grown in greenhouse and the leaves, stem and root proteome

mixtures revealed the differentially expressed peptides (Menossi et. al. 2009).

Furthermore, the proteomic approach was used study the protein differential

expression against bacterial infection by X. albilineans which causes Leaf Scald

disease in sugarcane (Freddy, 2011). The objective was to identify and characterize

the differentially expressed proteins in sugarcane infected by Leaf Scald disease.

Comprehending the different conditions related to resistance and susceptibility in

different sugarcane clones will possibly allow the monitoring of the reaction of

sugarcane to leaf scald within time and elucidate molecular aspects of the expression

of resistance. The information which is generated by the proteome analysis could be

integrated with sugarcane genomic data to improve the understanding of the nature of

the sugarcane and the abiotic and/or biotic form of interaction. In addition, it may

perhaps be possible to utilize some of the proteome information acquired to develop

molecular candidate markers that could be applied in marker-assisted selection

(Freddy, 2011).

2.12.2 Assessment of the protein expression in Escherichia coli

For recombinant protein expression in higher eukaryotes which have large genomes

like plants seems to be often difficult and purification is intricate. Thus expression

levels tend to be lower and the proteins expressed be liable to misfold and aggregate.

Escherichia coli (E. coli) is the most extensively and earlier prokaryotic expression

system for recombinant protein production (Frommer and Ninnemann 1995). The

relatively simple system, rapid growth rate and comparatively lower cost has been a

favourite selction for transformation studies. The available commercially inducible

cloning vectors are compatible with E. coli with an important limitation for obtaining

large amounts of soluble and functional proteins. Frequently proteins are accumulated

in the inclusion bodies within the bacteria (Yin et. al. 2007). Many experiments have

been carried out in E. coli for rapid subcloning of genes and expression of protein and

its solubility levels. Experimental approaches have been practised to avoid the


40

formation of these aggregates with the use of cold (Qing et. al. 2004, Le and

Schumann 2007), higher concentration of molecular chaperones (Mogk et. al. 2002),

lower IPTG concentration (Winograd et. al. 1993), IPTG induced in the late log phase

(Galloway et. al. 2003), and lower growth temperature of IPTG induced cultures

(Schein and Noteborn 1988; Vera et. al. 2007). The advantages of using E. coli as

expression system provide fast growth kinetics. This means the culture with a 1/100

dilution od starter starter culture reach the stationary phase in less time period

(Sezonov et. al. 2007). Secondly the high cell density cultures can be easily achieved.

The exponential growth in the medium leads to the densities near that number 1 ×

1013

viable bacteria/ml (Lee, 1996; Shiloach and Fass 2005). Thirdly the rich complex

media is widely available and reasonably priced. Lastly, the transformation with the

foreign DNA is quick and effortless. The plasmid transformation of E. coli can be

executed in as fewer than 5 min (Pope and Kent 1996).

Various transcription factors have been expressed in the E. coli for different stress

responses under the stress condition. The AP2/ERF, MYB, MYC, bZIP, HSF, NAC,

WRKY and C2H2 zinc-finger transcription factors have shown essential role under

biotic and abiotic stress conditions (Singh et. al. 2002; Vincour and Altman 2005;

Yamaguchi-Shinozaki and Shinozaki 2006). The SbDREB2A transcription factor

showed overexpression of plant stress towards tolerant functional gene grown in E.

coli cells (Gupta et. al. 2010), a group 3 LEA protein from soybean have also shown

salt stress tolerance in E. coli (Liu and Zheng 2005), also expression of phytochelatin

synthase gene in E. coli showed better protection towards the heat, salt, pesticide,

heavy metal and UV stress conditions (Chaurasia et. al. 2008). The better tolerance

towards salt, PEG and heavy metal stress by MuNAC4 in E. coli cells supporting its

stress responsive nature (Pandurangaiah et. al. 2012).

2.12.3 pET vectors for protein expression studies

The pET system is the most powerfµl system developed for the cloning and

expression of recombinant proteins in E. coli. Target genes are cloned in pET

plasmids under control of strong bacteriophage T7 transcription and translation

signals and expression is induced by providing a source of T7 RNA polymerase in the


41

host cell. T7 RNA polymerase is so selective and active that almost all of the cell‟s

resources are converted to target gene expression. Further, the desired product can

comprise more than 50% of the total cell protein a few hours after induction. The pET

vectors were originally constructed by Studier and colleagues (Studier and Moffatt

1986; Rosenberg et. al. 1987; Studier et. al., 1990). The unique characters like

presence of fusion tags, antibiotic resistance, ease in cloning and diversity in bacterial

host selection helped prioritize the used of pET expression vector for protein

expression. The pET-29a+ vectors was selected for our expression studies which

carried an N-terminal S•Tag™

/thrombin configuration plus an optional C-terminal

His•Tag®

sequence

2.12.4 In silico attributes of plant MYB proteins

In silico analysis of protein sequences have recently been an add on in the research

community so as to comprehend the isolated proteins thoroughly and annotate the

sequences appropriately. Advantages of this computational analysis have supported

many studies carried out in vitro. The amalgamation of the two advanced techniques

have unbolt the doors for better understanding and predictions of a particular gene

product. Many transcription factor proteins have been targeted for such analysis,

focusing here the MYB transcription factors.

Putative 218 MYB proteins sequences isolated from wheat included 36 R2R3-type

MYBs and were analyzed for understanding their complete open reading frames and

the motifs flanking the MYB domain were evaluated using the MEME web server (Cai

et. al. 2012). The stress responsive elements like MYB associated with various

functions such as plant development, hormonal regulation and response to stress were

identified from rice and Arabidopsis using bioinformatical application (Ibraheem et.

al. 2012). Accessibility of Plant Transcription Factor Database has generated an

attainable prospect to distinguish the transcription factor/regulator genes in various

plants under different abiotic stresses (Kumari et. al. 2013). Genes related to MYB

transcription factors from 237,954 sugarcane and 110,000 eucalyptus ESTs during the

abiotic stress were examined with Arabidopsis and rice ESTs by splicing pattern

comparative analyses and other computational methods (Soares-Cavalcanti et. al.


42

2009). Lately, the simulated 3D structures of RTBP1 and NgTRF1 proteina were

docked with template DNA of TTTAGGG conserved sequence motif using

HADDOCK web server. The I-TASSER protein work bench provided 3D structures

of these proteins as well as the different parameters to characterize the proteins

revealing good physicochemical properties, structure, dynamics and binding mode

(Mukherjee et. al. 2015). Study conducted to identify the conserved microRNAs

(miR165, miR828a and miR828b) using the computational approaches in cotton were

likely to be MYB-related proteins involved in abiotic stress response (Boopathi and

Pathmanaban 2012). Bioinformatical search was carried out to discover the novel

microRNAs regulated in sugarcane plants subjected to waterlogging stress. Similar

microRNAs in relation with sorghum genome were identified in sugarcane showing

15 % for stress response (Khan et. al. 2014).

Proteins that are differentially expressed in the duration of these stress conditions may

be used in selection and development of new crop varieties with improved drought or

salinity tolerance, attending the growing demand for varied renewable energy sources.

However, further investigation to identify more functional proteins and validation of

their functions to ascertain their involvement in producing resistant varieties.

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