Adama Science And Technology University

An Overview of WRKY Transcription Factors in the Regulating of Abiotic and

Biotic Stresses in Cotton (Gossypium spp.)

A Ph.D. Seminar I Submitted To

Department of Applied Biology
School of Applied Natural Science
By
Kasahun Amare
Advisor: Geleta Dugassa (Ph.D.)

February 2022
Adama, Ethiopia
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 Presentation Outlines
1. Introduction
2. Origin, structure and domain of WRKY TFs
3. WRKY TFs in Gossypium spp.
4. Role of WRKY TFs
4.1. Abiotic stress response
a) Drought response
b) Salinity response
c) Temperature response
d) Oxidative response
4.2.Biotic stress response
5. Conclusions

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 1. INTRODUCTION
• Gossypium spp. belongs to genus Gossypium, family Malvaceae, consists > 50
species (Wendel and Grover, 2015).

• Gossypium arboreum, and Gossypium herbaceum are diploid (2n = 2x = 26).

• Gossypium hirsutum, and Gossypium barbadense are tetraploid (2n=4x=52)

(Boopathi et al., 2015).
• Cotton is cash crop around the world and provides:
• cotton lint for textile industry,
• cottonseed oil for culinary purposes, and
• protein-rich oil cake residues for live-stock(Ahmad and Hasanuzzaman, 2020).

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 Cont.…
• Because of its monetary significance, it is cultivated in more than 100 countries,
with major share from:
Pakistan
• China (6,423,000 tons),
Brazil
• India (6,162,000 tons), China

• United States (3,181,000 tons), USA

• Brazil (2,341,000 tons), and

• Pakistan (980,000 tons) (Shahbandeh, 2021).

India

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 Cont.…
• On the other hand, its production is constrained by:

• Unsuitable environmental situations / abiotic stress and

• Biotic stresses (Boopathi et al., 2015; Gull et al., 2019).

• To combat such conditions, plants in the growth and development process

mobilize a variety of intrinsic mechanisms.

• Numerous studies indicate that the protective mechanisms are regulated by the
expression of stress genes.

• These stress genes are known to be regulated at the transcription level (Heidel-
Fisher et al., 2018).

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 Cont.…
• TFs are present in most eukaryotes while others are lineage-specific (Yamasaki,
2016).

• In plants, TFs are encoded by 10% of the genes (Gonzalez, 2016).

• WRKY(Tryptophan, Arginine, Lysine, Tyrosine) TFs are one of the largest families
of transcription regulators that are found exclusively in plants (Wani et al., 2021).

• Numerous studies have examined the role of WRKY family from model plants to
crops and other species (Li et al., 2020).

• WRKY TFs have various functions(Shi et al., 2014; Sun et al., 2015).

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Figure 1: WRKY function in response to different stresses

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 2. Origin, Structure and Domains of WRKY TFs
•WRKY genes seem to originate in early eukaryotes and are widely
distributed in plants(Zhang and Wang, 2005).
•The first cDNA WRKY protein, SPF1, was cloned from a sweet potato
(Ipomoea batatas) that binds to the SP8a (ACTGTGTA) and SP8b
(TACTATT) sequences(Ishiguro and Nakamura, 1994).
•WRKY proteins have since been identified experimentally from various plants
•Wild oats (Avena fatua) (Rushton et al., 1995),
•Parsley (Petroselinum crispum) (Rushton et al., 1996),
•Arabidopsis thaliana (de Pater et al., 1996),
•Barley (Hordeum vulgare) (Sun et al., 2003) and
•Cotton (Gossypium arboreum) (Xu et al., 2004), etc.

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 Cont.…
• WRKY TFs varies depending on plant species, for example,
Solanum lycopersicum 81
Arabidopsis thaliana 90
Gossypium arboretum 111
Oryza sativa 128
Sorghum bicolor 134
Zea mays 161
Triticum aestivum 171
Gossypium raimondii 219
Gossypium hirsutum 238
Glycine max 296

etc. were discovered (Tian et al.,2020).

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 Cont.…
• The structures of the WRKY TFs are divergent; and consists of two parts:

1. The N-terminal DNA binding domain (DBD) ends to contain the WRKYGQR
amino acid sequence and

2. The C-terminal zinc finger motif (Phukan et al., 2016).

• The DBD sequence of WRKY is based on the hepta-peptide WRKYGQK motif,

• But in a few WRKY proteins, the WRKY a.a sequences replaced by WRRY,
WSKY, WKRY, WVKY, or WKKY motifs (Xie et al., 2004; Hung et al., 2004).

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 Cont.…
• Given the number of WRKY domains and the pattern of the zinc finger motif,
WRKY TFs from can be characterized in three groups (Rahaie et al., 2013).

• However, other scholar categorizes into four groups:

I (two WRKY DBDs),

 II (single DBD with different C2H2 zinc fingers),

 III (single DBD with C2HC zinc fingers), and

 IV (incomplete WRKY domain without zinc fingers) (Xie et al., 2005).

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 Cont.…
• Group I members usually contain two WRKY domains,

• While most proteins with one WRKY domain belong to Group II.

• Group III proteins also have a single WRKY domain; however, the pattern of zinc
finger motif is unique/ different.

• Group II was further subdivided into five subgroups(Li et al., 2020) (Figure 2&3).

• Furthermore, some WRKY proteins contain a

• glutamate enrichment domain,
• a proline enrichment domain, and
• a leucine zipper structure (Chen et al., 2012).

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Figure 2: WRKY domain and zinc finger motif

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 3. WRKY TFs in Gossypium spp.
• TFs for three Gossypium sp. were generated from the Plant TF database.
Gossypium spp. TFS WRKY

Gossypium hirsutum 5,022 238

Gossypium raimondii 4,894 219

Gossypium arboretum 2,532 111 (Tian et al., 2020).

• 116 WRKY TFs in Gossypium raimondii (Dou et al., 2014),

• 239 GhWRKY TFs in Gossypium hirsutum and

• With three incomplete WRKY domain (GhWRKY27,238 & 239) (Gu et al., 2018).

• 15 GhWRKY IIa were scattered on chromosomes 5, 6 & 7 (Gu et al., 2018).

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 4. Roles of WRKY TFs in Stress Response
• The process of plant adaptation to environmental stresses is controlled by the
orchestration of complex molecular networks regulated by TF proteins (Yadav et
al., 2018).

• WRKY works in diverse biological functions in plants including Gossypium spp.

Plant defense response (Schluttenhofer and Yuan, 2015).

Abiotic stress responses, (Cheng et al., 2021).

Some WRKY involve in hormone signaling (Rahaie et al., 2013).

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 4.1 The Roles of WRKY TFs in Abiotic Stress Responses
• Abiotic factors are non-living chemical and physical elements in the environment
that affect organisms.

• Plants established general defense against abiotic stress through a complex

regulatory network that includes: Upstream signaling molecules
• Stress hormones,
• Reactive oxygen species,
• Polyamines,
• Phytochromes, and calcium
• Downstream regulation elements, TFs (Jiang et al., 2016; He et al., 2018).

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Figure 3: Abiotic stress signaling pathways(Khan et a., 2018).

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 A. Drought Response
Drought is a meteorological term and is commonly defined as a combined interplay of
• reduced rainfall, decreasing ground water table,
• limiting water availability with rise in temperature

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Figure 4: Effects of drought stress on cotton and their responses (Source:ullah et al.,2017).
 Cont...
• In cotton production, drought is a key limiting factor (Li et al., 2017).

• The yield is severely restricted by drought stress (Wang et al., 2019).

• It has been reported:

Seed yield decrease by 42% and

The biological yield decrease by 55% (Wang et al., 2019).

• Genes and TFs responsible for stress tolerance in crop plants were
identified through molecular and biochemical studies (Yadav et al.,
2018).

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 Cont.…
• Usually regulated by multiple genes, including those encode WRKY TFs (Pinto et
al., 2010; Dou et al., 2014; He et al., 2018).

• Members of the WRKY family play an important role in various stress reactions (Li
et al., 2020).

• Research has shown that over-expression of AtWRKY57 can increase the

drought tolerance of Arabidopsis (Jiang et al., 2016).

• The GhWRKY1-like from upland cotton positively regulate drought tolerance by

involving in ABA biosynthesis(Hu et al., 2021) .

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 Cont.…
• GhWRKY17 was found to be induced after exposure to drought, salt, H 2O2, and ABA
(Yan et al., 2014).

• ABA is synthesized to induce stomatal closure in order to prevent water loss by

transpiration (Schroeder et al., 2001)

• GhWRKY17 modulated the increased sensitivity of plants to drought by reducing ABA

levels, and

• Transcript levels of ABA-inducible genes, including AREB, DREB, NCED, ERD, and
LEA, were markedly suppressed under drought and salt stress conditions (Yan et al.,
2014).
• GhWRKY33 and GhWRKY41 enhances drought and salt stresses by regulating
stomatal movement (Chu et al., 2015; Wang et al., 2019).
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 Cont.…
• GhWRKY68 may mediate salt and drought responses by modulating ABA
content and enhancing the transcript levels of ABA-responsive genes ( Jia et
al., 2015).

• GhWRKY59, response to drought in cotton through phosphorylation by MAPK

cascade consisting of GhMAP3K15, GhMKK4, GhMPK6 (Li et al., 2017) .

• GhWRKY59 binds directly to the promoter of GhDREB2 and regulates drought-

responsive gene expression.

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Figure 5: A model of the Mitogen-Activated Protein (Source: Li et al., 2017).

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 B. Salt Stress Response
• Salt is an environmental determinant that restricts plant growth, lowers crop yield,
and is an increasingly serious problem worldwide (Yang and Guo, 2018).

• Salt stress affects plants in different ways: osmotic stress, ionic stress, and
particularly oxidative stress (Yang and Guo 2018).

• The expression rates of genes associated with numerous biological processes

and signaling pathways are significantly affected by salt stress (Wang et al.,
2020)..

• Compared to other model plants, a limited variety of salt tolerance genes have
been identified in cotton (Wang et al., 2020).

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 Cont.…

• Complex molecular mechanisms of the salt stress response in cotton are the
activation of various transcription factors like MYB, WRKY, AP2, and cell wall
modification and are highly active in response to salt stress (Wang et al., 2020).

• Salt-sensitive WRKY genes from cotton have been identified and characterized
(Shi et al., 2014; Yan et al., 2014; Chu et al., 2016).

• Transgenic plants in the vegetative stage, wild type(WT) and overexpressing(OE)

plants were irrigated with salt water (200 mM) for a month to observe
GhWRKY68.

• The OE plants showed severe growth retardation and serious leaf curling and
chlorosis compared with WT plants after NaCl treatment.
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 Cont.…

• Approximately 40% of the OE plants survived the high-salinity conditions, with the
survival rate lower than that of WT plants.

• The over-expression of GhWRKY68 might confer reduced tolerance to salt stress

in transgenic plants during seed germination and in the vegetative stage (Jia et al.,
2015).

• Prior to this report, GhWRKY17 transcript rapidly increased upon NaCl treatment,
reaching its maximum accumulation (2.5-fold induction) at 6h and gradually
diminishing thereafter (Yan et al., 2014).

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 Cont.…
• GarWRKY17 and GarWRKY104 can enhance salt tolerance during different
developmental stages (Fan et al., 2015).

• GarWRKY5 in Gossypium aridum is involved in salt stress response by the JA or

SA signaling (Guo et al.,2019).

• GarWRKY5-overexpressing exhibited higher activities of superoxide dismutase

and peroxidase under salt stress (Guo et al., 2019).

• GhWRKY6 increases salt tolerance through binding of GhWRKY6 to three W-

boxes within a 1Kb upstream of the RAV1 gene (Li et al., 2019).

• Upland cotton variety LMY37 and ZM12 showed strong reactions to salt stress
due to the overexpression of GhWRKY6 proteins(Zhang et al., 2021).

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 C. Temperature Stress Response
• Temperature levels beyond an organism’s optimal tolerance range are regarded
as major abiotic stress.

• Thus, devising strategies to protect plant cells from damages caused by

temperature is necessary to boost agricultural production (Ohama et al., 2017).

• The WRKY family is known to be involved in the high-temperature response

• Regulate gene expression through multiple pathways and at different levels, (Zhou
et al., 2008).

• Different studies have shown that WRKY TFs can respond positively to
temperature stress.

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 Cont.…

• For example,

• CaWRKY40 in pepper participates in the response to high-temperature stress,

while the loss of this TF reduces this tolerance (Dang et al., 2013).

• The constitutive over-expression of AtWRKY25 and AtWRKY26 increased

resistance to high-temperature stress (Fu and Yu, 2015).

• WRKY25, WRKY26, and WRKY33 proteins functionally interact and play

synergistic roles in plant thermo-tolerance (Li et al., 2020)

• A similar result was found for GhWRKY39, expressed in upland cotton (Shi et al.,
2014).

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 Cont.…
• The over-expression of TaWRKY30 led to high-T0 tolerance (Zhu et al., 2013).

• In rice HSP101 promoter-driven over-expression of OsWRKY11 led to enhanced

heat and drought tolerance (Wu et al., 2009).

• However, WRKY may play a negative role in response to high-T0 tolerance;

• For example,

• The heterologous expression of HaWRKY6 in Arabidopsis could significantly

reduce their tolerance to high T0 (Raineri et al., 2015).

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 D. Oxidative Stress Response

•Oxidative stress is a complex chemical and physiological phenomenon.

•Develops as a result of overproduction and accumulation of ROS)
(Demidchik,2015),
•Due to the disruption of various metabolic pathways, such as respiration and
photosynthesis (Pessarakli, 2019).
•Many biological processes are controlled by ROS, which includes:

Programmed cell death,

Biotic and abiotic stress responses, and

Plant growth and development (Xu et al., 2019).
•Plants prevent damage by stimulating multiple stress-related genes that are
associated with the antioxidant defense system ( Priya et al., 2019).
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 Cont.…
• These are governed by different genes groups such as
1. Genes involved in the defense of cell membranes, such as
• osmotic protectants,
• radical scavengers, and
• detoxification enzymes;
2. Genes involved in signaling cascades and transcription control, e.g.
Mitogen-activated protein kinase (MAPK),
Phospholipases,
Calcium-dependent protein kinase (CDPK),
Phospholipases and transcription factors (TFs) and
3. Genes involved in the uptake and transport of ions (Ciarmiello et al., 2011).

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 Cont.…
• ROS network-mediated signal pathway enhances the mechanism of fiber
development and regulation of abiotic stress in cotton (Xu et al., 2019).

• 515 in Gossypium hirsutum, 261 in Gossypium raimondii, and 260 in Gossypium

arboreum ROS regulating genes were reported.

• High no of ROS genes suggested a complex ROS network for enhancing abiotic
stress tolerance (Xu et al., 2019).

• Overexpression of GhWRKY39-1, GhWRKY44 was enhancing resistance by

inhibiting the accumulation of pathogen-induced ROS (Shi et al., 2014).

• GarWRKY5 over-expression in Arabidopsis resulted in increased activities of

anti-oxidative enzymes to scavenge the ROS (Guo et al., 2019).

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 Cont.…
• GhWRKY6 were also tested in the oxidative stressors of malondialdehyde and
H2O2 in transgenic and WT plants, and

• Then a greater accumulation of malondialdehyde and H2O2 in WT was observed

compared to transgenic lines.

• From the result it became evident that WT plants were seriously affected by ROS,
while the over-expressing GhWRKY6-like transgenic lines were protected
compared to WT plants (Ullah et al., 2018).

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Figure 6: Antioxidative Defense (Source: Mahmood et al., 2020).

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 4.2. Biotic Stress Response
• Biotic stress affects all phonological stages of plant development.

• Biotic stress includes attacks by pathogenic bacteria, fungi, and viruses (Jiang et
al., 2016).

• Various defense mechanisms have evolved in plants to fight microbial infections.

• Transcription factors are central to this process.

• Large number of defense genes are regulated at the transcriptional level in

response to pathogen infection (Eulgem and Somssich, 2007; Jiang et al., 2016).

• WRKY proteins interact with other TFs to regulate the defense reactions of
plants (Eulgem and Somssich, 2007; Jiang et al., 2016).

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 Cont.…
• Multiple WRKYs can confer resistance to multiple bacterial or fungal pathogens.

• AtWRKY52, works in conjunction with RPS4 to create resistance to the fungal

and bacterial pathogens

• Colletotrichum higginsianum

• Pseudomonas syringae (Narusaka et al., 2009).

• AtWRKY52 shows a nuclear interaction with bacterial effector PopP2
offers immunity to Ralstonia solanacearum (Deslandes et al., 2002).
• AtWRKY52 / RRS1,
• AtWRKY16 / TTR1, and
• AtWRKY19, activate defense-related genes (Rushton et al., 2010).

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 Cont.…
• AtWRKY16 and AtWRKY19 are involved in the defense-related ETI signaling
pathways (Chi et al., 2013).

• Mutations in WRKY70 and WRKY33 increased the susceptibility of Arabidopsis to

Botrytis cinerea (Birkenbihl and Somssich, 2011).

• WRKY40, WRKY33, WRKY53, WRKY22, WRKY11, WRKY15, and WRKY60 are

induced with wounding in Arabidopsis (Cheong et al., 2002).

• WRKY TFs were expressed differently in fungal infection in cottonseeds.

• WRKY75 and WRKY40 were up-regulated in pericarp and seed under infection.

• WRKY6, WRKY41, WRKY50 and WRKY53 were down-regulated in the pericarp

(Bedre, 2016).
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 Cont.…
• The GhWRKY40 was up-regulated in response to infection with Ralstonia
solanacearum.
• However, the overexpression enhances the susceptibility of tobacco plants to
Ralstonia solanacearum (Wang et al., 2014).
• Over-expression of GhWRKY39 in Nicotiana benthamiana conferred greater
resistance to bacterial and fungal pathogen infections (Shi et al., 2014).
• GhWRKY70 expression was induced by Verticillium dahlia infection, SA, and Me
JA treatment (Xiong et al., 2019).
• In particular, JA-response-associated genes were up-regulated and SA-related
genes were down-regulated in GhWRKY70 virus-silenced cotton plants.

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 Cont.…
• GbWRKY1 is regulated by the SA, JA, and ET pathways and is primarily involved
in resistance to Verticillium dahliae via the ET pathway (Zhang et al., 2019).

• AtWRKY70 was likely involved in brokering SA-JA crosstalk and that AtWRKY70
is activated by SA and suppressed by JA (Liet al., 2020).

• In cotton, GhWRKY7, GhWRKY27, GhWRKY31, GhWRKY50, GhWRKY56,

GhWRKY59, GhWRKY60 and GhWRKY102 were significantly upregulated when
treated with SA and JA, and these genes implied potential functions in SA and JA
signaling pathways (Dou et al., 2016).

• This provides evidence of the positive regulatory functions of GhWRKY resistance

to pathogenic infections (Li et al., 2015).

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 5. CONCLUSIONS
• TFs play an important role in plant biotic and abiotic stress responses

• 58 TF families have been identified to be involved in the plant stress response.

• WRKY TFs are the most prominent and have been studied in various plant
species including Gossypium spp.

• The WRKY-TFs are identified as molecular stress regulators to combat

• biotic (bacteria, fungi, and viruses, etc.) and
• abiotic (drought, salinity, temperature, oxidation, etc.) stresses.
• It is proposed to investigate in the future:
 How these WRKY TFs are networked with other regulatory elements to resist external
stimuli and
 Understand their involvement in metabolic activities.

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 Acknowledgements

•First, I would like to thank my advisors, Dr. Geleta Dugassa.

•With extremely humble greetings, I bow my head to the respected instructors
• Dr. Mulugeta Kebede and
• Dr. M. Naimuddin,
• For their support and valuable comments during the title construction.
• Words cannot recognize the contribution of my beloved friends
• Wondimu Huluka, PhD student at ASTU,
• Endalachew Baye, Selale University,
• For their critical suggestions in writing.

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 The End

Thank you

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