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dr Jadwiga Helena Szczegielniak 25.04.2019 Instytut Biochemii i Biofizyki PAN

Description of scientific achievements

1. Name and surname: Jadwiga Helena Szczegielniak 2. Education history. 1999 PhD Biochemistry, Institute of Biochemistry and Biophysics PAS, Warsaw,

Thesis title: Calcium and phospholipids-dependent protein kinase from maize

seedlings.

Advisor: Prof. Grażyna Muszyńska

Reviewers: Prof. Nikodem Grankowski (Maria Curie Skłodowska University in Lublin)

and Doc. Dr hab. Katarzyna Nałęcz (Nencki Institute of Experimental Biology, PAS)

1979 M.Sc. Nicolaus Copernicus University, Toruń, Thesis title: Development of method

for measuring N-sulphaminidase activity.

Advisor: Prof. Piotr Masłowski

3. Employment history.

10.1983 - 08.1986 biologist, Institute of Biochemistry and Biophysics, PAS, Warsaw

10.1986 - 11.1993 biologist, Institute of Biochemistry and Biophysics, PAS, Warsaw

12.1993 - 06.2000 research assistant, Institute of Biochemistry and Biophysics, PAS,

Warsaw

07.2000 - 04.2014 assistant professor, Institute of Biochemistry and Biophysics, PAS,

Warsaw

since 2014 biologist, Institute of Biochemistry and Biophysics, PAS, Warsaw

4. Selected scientific achievements. (Achievement indication* according to art. 16 act. 2 of legislation of March 14, 2003 on the scientific degrees and scientific title and degrees and title in Art (Journal of Law No 65, item. 595, as amended). * Statements of all co-authors determining the individual contribution of each of them in the

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creation of individual works can be found in “Deklaracje współautorów”. a) Title of scientific artistic achievement:

The role of Zea mays calcium-dependent protein kinase (ZmCPK11)

in response to abiotic stresses

b) Publications included in the scientific achievement: 1. Szczegielniak J, Klimecka M, Liwosz A, Ciesielski A, Kaczanowski S, Dobrowolska G, Harmon AC, Muszyńska G (2005) A Wound-Responsive and Phospholipid-Regulated Maize Calcium-Dependent Protein Kinase. Plant Physiol 139: 1970-1983 2. Klimecka* M, Szczegielniak* J, Godecka L, Lewandowska-Gnatowska E, Dobrowolska G, Muszyńska G (2011) Regulation of wound-responsive Calcium-Dependent Protein Kinase from maize (ZmCPK11) by phosphatidic acid. Acta Biochim Polon 58: 589-595 *equal authors contribution 3. Szczegielniak*J, Borkiewicz L, Szurmak B, Lewandowska-Gnatowska E, Statkiewicz M, Klimecka M, Cieśla J, Muszyńska G (2012) Maize calcium-dependent protein kinase (ZmCPK11): local and systemic response to wounding, regulation by touch and components of jasmonate signaling. Physiol Plant 146: 1-14 *corresponding author 4. Lewandowska-Gnatowska E, Polkowska-Kowalczyk L, Szczegielniak J, Barciszewska M, Barciszewski J, Muszyńska G (2014) Is DNA methylation modulated by wounding-induced oxidative burst in maize? Plant Physiol Biochem 82: 201-208 5. Borkiewicz L, Polkowska-Kowalczyk L, Cieśla J, Sowiński P, Jończyk M, Rymaszewski W, Szymańska KP, Jaźwiec R, Muszyńska G, Szczegielniak* J (2019) Expression of maize Calcium Dependent Protein Kinase (ZmCPK11) improves salt tolerance in transgenic Arabidopsis plants by regulating sodium and potassium homeostasis and stabilizing photosystem II. Physiol Plant doi.org/10.1111/ppl.12938 *corresponding author

c) Discussion of the scientific work and the results and their possible use:

The role of Zea mays calcium-dependent protein kinase (ZmCPK11)

in response to abiotic stresses

Introduction

A common mechanism regulating signaling pathways is the post-translational,

reversible phosphorylation of proteins, in which both kinases and protein phosphatases are

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involved. A protein kinase activated by calcium ions (Ca2+) and stimulated by some

phospholipids was identified in maize seedlings (Muszyńska et al. 1993). This kinase

regulation by lipids resembled animal protein kinase C (PKC) involved in the signal

transduction pathways of many physiological processes and disease (Mellor and Parker

1998). The lipid-stimulated protein kinase activity, referred by authors to "PKC-like", has also

been identified in zucchini, potato, in wheat cells and in maize seedlings (Chandok and

Sopory 1998). However, it has not be able to possible to document the existence of PKC in

plants by means of molecular cloning. Our attempts to clone PKC in maize have also failed.

Therefore, functional PKC analogs have been sought in plants. The only known enzyme that

could act as PKC in plants was Ca2+-dependent protein kinase - CDPK/CPK (Calcium-

Dependent Protein Kinase/Calmodulin-Domain Protein Kinases), because the activity of

some CDPK was regulated by phospholipids. These included oat CDPK, activated by

phosphatidylinositol (PI), lysophosphatidylcholine (LysoPC) and endogenous lipids from soy

(Schaller et al. 1992), AtCPK1 from Arabidopsis, stimulated by lysophosphatidylcholine (PC)

and PI (Harper et al. 1993) and DcCPK1 from carrot, stimulated by phosphatidylserine (PS), PI

and phosphatidic acid (PA) (Farmer and Choi 1999). Therefore, the main goal of our research

was the classification and characterization of Ca-dependent and phospholipid-stimulated

protein kinase, which was isolated from maize seedlings in a laboratory managed by prof.

Grażyna Muszyńska. We were interested in whether in plants PKC exists and whether kinase

we study is a classic PKC or CDPK kinase, whose activity is regulated by PKC activators? The

protein kinase from maize seedlings has been purified to a state of near homogeneity. Its

biochemical and immunological characteristics indicated that the tested kinase is definitely a

CDPK and was named ZmCPKp54 (Szczegielniak et al. 2000). The results of these studies

were the basis of my doctoral dissertation. Getting to know the genome of Arabidopsis

thaliana (Cheng et al. 2002), and then the genomes of other plants confirmed that PKC does

not exist in plants. On the other hand, present in plants the Ca2+ dependent kinases - CDPKs,

there are not exist in animals or fungi.

CDPK are involved in the earliest stages of the response to intracellular and

environmental signals. One of the first effects of the signal is a transient increase in the

concentration of Ca2+ in the cytoplasm, which act as a second messenger. Second

messengers, in addition to Ca2+, also include structural membrane components -

phospholipids, including PA, which is rapidly formed by the hydrolysis of PC in response to a

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signal (Wang et al. 2006, Kudla et al. 2018). Transient changes in Ca2+ concentrations in the

cytoplasm are referred to as "calcium signatures". “Calcium signatures” vary depending on

the type of signal, its intensity, location and duration (Kudla et al 2018). The decoders of

information recorded in the "calcium signatures" are calcium ion sensor proteins, and

among them, unique to plants, green algae, protozoa and oomycetes, CDPK kinases. These

kinases are encoded by multi-gene families, e.g. in Arabidopsis thaliana there are 34 genes

coding for CDPK, in rice 29, in potato 23, in maize 35 and 40 (Simeunovic et al. 2016,

Gromadka et al. 2018). A typical CDPK is a monomeric protein composed of N- and C-

terminal variable domains and three conserved functional domains: a catalytic serine-

threonine kinase domain, a junction domain and regulatory domain, similar to calmodulin

with four Ca2+ binding motifs called "EF-hand". Due to this structure, CDPKs are particularly

interesting proteins because they represent the type of sensors of changes in Ca2+

concentrations due to the regulatory domain and simultaneously the effectors, via the

catalytic domain of the kinase. Ca2+ bind to a calmodulin-like domain, alter protein

conformation and activate a kinase that, by phosphorylating the appropriate proteins,

induces subsequent events in the signal transduction pathways. Currently, CDPKs are one of

the most intensively studied kinases.

Studies of recent years indicate that CDPKs are important both in the stress signal

transduction pathways as well as in pathways activated during plant growth and

development (Simeunovic et al. 2016, Shi et al. 2018, Bredow and Monaghan 2019). For

example, CDPK from Arabidopsis: AtCPK2, AtCPK17, AtCPK20 and AtCPK34 are mainly

expressed in pollen and are involved in pollen tube growth and elongation. Two other

kinases, AtCPK11 and AtCPK24, also regulate the elongation of the pollen tube by regulating

the K+-channels. In addition, studies on individual CDPK isoforms have shown that

overproduction of one protein is able to increase or decrease plant tolerance to abiotic

stresses or resistance to microorganisms and pathogens (Xu and Huang 2017, Shi et al. 2018,

Aldon et al. 2018, Bredow and Monaghan 2019). The overexpression of OsCPK21 in rice,

increases ABA-induced gene expression and salinity, resulting in a higher tolerance of plants

to salinity (Asano et al. 2011), whereas overexpression of OsCPK12 in rice also increased

tolerance to salinity, but by reducing ROS accumulation. The OsCPK4-transgenic plants were

better protected against cell membrane damage caused by oxidative stress (Simeunovic et

al. 2016) as well as being more resistant to Magnaporthe oryzae fungus (Aldon et al. 2018).

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Heterologous expression of maize three CDPK genes in Arabidopsis has identified ZmCPK1,

as a negative regulator of cold stress signaling (Weckwerth et al. 2015), ZmCPK4, as a

positive regulator of drought stress tolerance (Jiang et al. 2013) and ZmCPK12, as a positive

regulator of drought stress tolerance (Wang and Song 2013).

Functional plant analysis showed that in Arabidopsis AtCPK4, AtCPK5, AtCPK6 and AtCPK11

positively regulate gene expression in response to bacterial flagellin and participate in

resistance to Pseudomonas syringae (Aldon et al. 2018). Many plant CDPKs was found to be

involved in phytohormone synthesis and signaling (Simeunovic et al. 2016, Xu and Huang

2017, Shi et al. 2018). For example, AtCPK28 is a key regulator of gibberellic acid (GA) and

jasmonic acid (JA) level and functions in JA-dependent developmental processes in stem

elongation and vasculature, which are independent of JA-dependent responses to stresses.

Several CDPKs have been reported to phosphorylate a key enzyme of the ethylene

biosynthesis - 1-aminocyclopropane-1-carboxylic acid synthase (ACS). AtCPK16

phosphorylates in vitro ACS7 in three sites, but the importance of these modification in vivo

is unknown. Other CDPKs as AtCPK4 and AtCPK11 play a positive role in ethylene

biosynthesis. AtCPK4 and AtCPK11 phosphorylate the C-terminus of ACS6 in vitro and

enhance the stability of ACS6. Loss-of-function a cpk4/cpk11 double mutant treated by ABA

produce less ethylene. In tomato, LeACS2 is immediately phosphorylated by two protein

kinases, LeCPK2 and MAPK after wounding. LeACS2 stability required phosphorylation by

both kinases (Shi et al. 2018). CDPKs in Arabidopsis (AtCPK3, AtCPK4, AtCPK6, AtCPK10,

AtCPK11 and AtCPK32) play a positive role in ABA-dependent physiological processes, such

as seed germination, post-germinate growth, stomata movement and plant tolerance to

stress (Simeunovic et al. 2016, Shi et al. 2018, Bredow and Monaghan 2019).

In addition, the growing number of identified CDPK substrates indicates the regulatory role

of these enzymes in many other physiological processes (Simeunovic et al. 2016, Bredow and

Monaghan 2019). In Arabidopsis, CDPKs such as AtCPK4, AtCPK5, AtCPK6, and AtCPK11 play

a key role in defence-induced ROS production. StCDPK4 and StCDPK5, close homologues of

AtCPK5/AtCPK6, can directly phosphorylate the NADPH oxidases StRbohB and StRbohC to

induce ROS production. Moreover, several CDPKs regulate the movement of stomata by

phosphorylation of ion channels. These include AtCPK21, which activates SLAC1 (Slow Anion

Channel 1) and its homolog, SLAH3 channels, in response to ABA and functions as a positive

regulator in ABA-induced stomatal closure. AtCPK8 plays a role in the movement of stomata

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by regulating the enzyme associated with the degradation of H2O2, catalase 3 (AtCAT3), in

ABA-dependent response to drought.

However, knowledge of the role of these kinases is still fragmentary. This is particularly true

for crop plants such as maize, one of the most important crops in the world.

Description of the publications

After obtaining the doctoral degree, I continued the study of Ca2+-dependent and

phospholipids-stimulated protein kinase, ZmCPKp54, identified as CDPK (Szczegielniak et al.

2000). I cloned and sequenced the cDNA encoding the studied kinase, and then, I focused

on the molecular characteristics of recombinant kinase and the search for its biological

role. The results are presented in the publication

Szczegielniak J, Klimecka M, Liwosz A, Ciesielski A, Kaczanowski S, Dobrowolska G, Harmon AC, Muszyńska G (2005) A Wound-Responsive and Phospholipid-Regulated Maize Calcium-Dependent Protein Kinase. Plant Physiol 139: 1970-1983

The cloning strategy consisted in purifying the kinase protein from maize seedlings to a state

of homogeneity based on a previously developed procedure with modifications

(Szczegielniak et al. 2000), followed by microsequencing of purified protein. The sequences

of the obtained peptides (homologous to CDPK sequences) and EST (Expressed Sequence

Tag) sequences from monocotyledon plants were used to design primers and amplify the

cDNA of the kinase by RT-PCR. This kinase, as the eleventh CDPK of maize, was named

ZmCPK11 (the former name of ZmCPKp54), and the sequence deposited in the

GeneBank/EMBL nucleotide database under number AY301062.2. ZmCPK11 has a typical

CDPK structure with three highly conserved domains (protein kinase domain, calmodulin-like

domain and junction domain) and N- and C-variable domains. The N-variable domain of

ZmCPK11 showed great similarity to the EST sequence from monocotyledones plants of

sorghum, barley and oat, while it did not show similarity to any CDPK from dicotyledonous

plants. This suggested that the similarity of N-variable CDPK domains is limited to narrow

groups of closely related plants. For in vitro experiments we have produced in the bacterial

system the ZmCPK11 protein recombined in fusion with S-glutathione transferase (GST) and

we performed its biochemical characteristics in terms of substrate specificity and regulation

by calcium and phospholipids. Recombinant ZmCPK11, like the native kinase (Szczegielniak

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et al. 2000), phosphorylated histone III-S and Syntide 2 peptide in a Ca2+ dependent manner,

although lower Ca2+ concentrations were required for phosphorylation of Syntide 2.

Phospholipids such as PA, PS and PI strongly stimulated the activity of ZmCPK11 in the case

of histone III-S phosphorylation, but not of the peptide substrate Syntide 2. Other lipids such

as PC, LysoPC, cardiolipin, diolein, phosphatidylethanolamine and n-dodecyl-β-D-maltoside

detergent did not stimulate ZmCPK11 activity. The obtained results indicated that the

stimulation of ZmCPK11 activity by phospholipids and the affinity of the enzyme for Ca2+

depends not only on the type of lipid, but also on the substrate. The autophosphorylation of

ZmCPK11 was also stimulated by Ca2+ and phospholipids such as PA and PS.

Analysis of expression showed the presence of the ZmCPK11 transcript in all the analyzed

maize organs (seeds, stems, roots, leaves), but the highest level of expression was in

seedlings. In response to mechanical wounding, ZmCPK11 is independently regulated at two

different levels of enzyme activation and synthesis of specific mRNA. Mechanical wounding

of maize leaves activated a 56 kDa Ca2+ dependent kinase, which the molecular weight

corresponded to the molecular weight of ZmCPK11. The ZmCPK11 expression also increased,

locally at the wounding site and systemically in the adjacent, uninjured leaf.

Summing up: the results obtained at this stage of the study led to the known of the cDNA of

the ZmCPK11 kinase and obtaining the functional protein ZmCPK11 in the bacterial system.

in vitro studies have shown that the recombinant ZmCPK11 in terms of substrate specificity,

regulation by Ca2+ and phospholipids is similar to the native kinase from maize seedlings. In

addition, we discovered that ZmCPK11 is involved in local and systemic response to

mechanical wounding.

Our studies have shown that the in vitro activity of both native and recombinant

ZmCPK11 kinase is stimulated several times by phospholipids such as PA, PS and PI

(Szczegielniak et al. 2000, 2005). However, it was not known whether lipids regulate CDPK

activity in vivo and the mechanism of this regulation was not known. Therefore, the next

stage of the research was to learn about the role of PA in regulating ZmCPK11 in the

wound signaling pathway. The results described below have been published in the

publication

Klimecka M, Szczegielniak J, Godecka L, Lewandowska-Gnatowska E, Dobrowolska G, Muszyńska G (2011) Regulation of wound-responsive Calcium-Dependent Protein Kinase from maize (ZmCPK11) by phosphatidic acid. Acta Biochim Polon 58: 589-595

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Treatment of maize leaves with exogenous PA induced in vivo the ZmCPK11 enzymatic and

transcriptional activity. This activation of ZmCPK11 coincided with the stimulation of activity

and the increased expression of ZmCPK11 after wounding (Szczegielniak et al. 2005). It

should be emphasized that in the cell membrane, after wounding, rapid hydrolysis of PC in

the phospholipase D pathway (PLD) forms the second messenger PA (Wang et al. 2006). We

observed that ZmCPK11 was also activated in vivo and increased its expression in maize

leaves treated with the PC, but later than after treatment with PA. Most likely, the delayed

response of ZmCPK11 in response to PC treatment was due to the time needed for PA

synthesis, and PC only indirectly affected the activity of ZmCPK11 through PA. This is

evidenced by the results of research in Arabidopsis showing that accumulation of PA after

being wounded is accompanied by a reduction in the amount of PC (Zien et al. 2001).

Considering the above facts, we assumed the participation of the PA produced in PLD

pathway in wound-induced activation of ZmCPK11. To verify the assumption of this

hypothesis, we used an inhibitor of PA synthesis in the PLD pathway - n-butanol (Krinke et al.

2009) and before the wounding the maize leaves were treated with n-butanol. The obtained

results indicated that that PA produced in the PLD pathway is involved in the regulation of

the in vivo activity of ZmCPK11 during wounding, since inhibition of the PLD signaling

pathway with n-butanol significantly inhibited the wound-induced activity of ZmCPK11.

Our research was also intended to clarify whether the stimulation of ZmCPK11 activity by

lipids is the result of the direct interaction of the enzyme with lipids. The binding studies of

ZmCPK11 with phospholipids were carried out using the "lipid binding assay" method using

nitrocellulose filters with immobilized lipids. Among the tested phospholipids, only PA 16: 0

and PA 18: 0 containing saturated acids, effectively bound to ZmCPK11. In contrast, PS and

PI, which also stimulated the activity of ZmCPK11 in vitro, did not bind to ZmCPK11 in vivo.

To determine which region of ZmCPK11 is responsible for binding to PA, we checked the

binding of PA to the entire enzyme, its inactive mutant (lysine 74 at the ATP binding site

changed to methionine) and truncated forms. Proteins for research were produced in

bacterial system and in the transient expression system in protoplasts from maize leaves.

The protein fragment containing the catalytic domain bound to PA 16: 0 and PA 18: 0 as did

the entire enzyme and its inactive form, whereas the ZmCPK11 fragment without the

catalytic domain did not bind lipids, which indicates that the region responsible for binding

the enzyme to PA is in the catalytic domain of ZmCPK11. Binding of the PA to the inactive

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mutant ZmCPK11-K74M suggested that the ATP binding site in ZmCPK11 is not responsible

for the binding of PA. In addition, only PA 16:0 and PA 18:0, in a concentration dependent

manner, stimulated the in vitro autophosphorylation of ZmCPK11. The degree of stimulation

of ZmCPK11 activity by PA was correlated with the level of autophosphorylation of the

enzyme, suggesting that phosphorylation of ZmCPK11 stimulates the enzymatic activity of

ZmCPK11.

Summing up: in this work we showed that in the activation of ZmCPK11 during mechanical

wounding, apart from Ca2+, is involved also another secondary messenger - PA produced in

the PLD pathway and that the stimulation of ZmCPK11 activity by PA may be the result of

direct kinase interaction with lipid.

The jasmonic acid (JA) biosynthesis pathway plays a key role in plant response to

mechanical wounding and attack of herbivores and necrotrophic pathogens nekrotroficznych

(Wasternack 2007). It has been shown that the majority of genes induced during wounding

are regulated by the octadecanoid pathway (JA biosynthesis). In addition, JA in the systemic

response, play the role of a long-distance signaling molecule transmitting information about

wounding to distant, undamaged places (Schilmiller and Howe 2005). Responses to

wounding, dependent and independent on JA, are regulated by the processes of reversible

phosphorylation of proteins (Leon et al. 1998).

Therefore, we wanted to answer the question whether ZmCPK11 also participates

in the systemic response to wounding at the level of enzymatic activity and whether it

participates in a JA-dependent wound signaling? In the work

Szczegielniak J, Borkiewicz L, Szurmak B, Lewandowska-Gnatowska E, Statkiewicz M, Klimecka M, Cieśla J, Muszyńska G (2012) Maize calcium-dependent protein kinase (ZmCPK11): local and Systemic response to wounding, regulation by touch and components of jasmonate signaling. Physiol Plant 146: 1-14

we have shown that 56 kDa Ca2+-dependent protein kinase is activated in vivo after just a

few minutes, both in wounded and unwounded maize leaves. This was the first report on the

participation of CDPK at the level of enzymatic activity in the systemic response to

wounding. To study the participation of ZmCPK11 in the JA-dependent wound signaling

pathway, we used the JA biosynthesis precursor - linolenic acid (LA, 18: 3) and JA methyl

ester (MeJA). Both compounds are components of the JA biosynthetic pathway and play an

important role in response to wounding. Applied exogenously, they induce in plants defense

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responses similar to those invoked by wounding (Farmer and Ryan 1992, Koch et al. 1999,

Halitschke et al. 2001). Treatment of maize leaves with LA or MeJA increased the level of the

ZmCPK11 transcript and activated the 56 kDa CDPK. We also checked the influence of touch

on the activity and expression of ZmCPK11, because the touch stimulates defense reactions

similar to those elicited by herbivores, despite the fact that it is weaker stress than

mechanical wounding and does not damage plants. The obtained results showed that the

touch also activates the 56 kDa CDPK and causes the increase in the level of the ZmCPK11

transcript. In the JA-dependent wound pathway, salicylic acid (SA) plays an antagonistic role.

SA and its acyl derivative (acSA, aspirin) suppress defense-related responses dependent on

JA (Leon-Reyes et al. 2010). Therefore, we examined the effect of acSA on wound-induced

activation of 56 kDa CDPK. Treatment of acSA maize leaves inhibited wound-induced

activation of 56 kDa CDPK. To demonstrate that 56 kDa Ca2+ -dependent protein kinase is

ZmCPK11 we decided to use the immunoprecipitation technique combined with analysis of

kinase activity in polyacrylamide gel (IP/in gel kinase activity assay) using antibodies specific

for ZmCPK11. Unfortunately, attempts to obtain specific antibodies recognizing native

ZmCPK11 failed. Therefore, to demonstrate the role of ZmCPK11 in response to stresses, we

used transgenic Arabidopsis thaliana plants with the introduced ZmCPK11 gene with the c-

Myc tag under the control of the cauliflower mosaic virus 35S promoter. The c-Myc tag

allowed to follow the activity of ZmCPK11 and its protein amount during stress conditions

using anti-c-Myc antibodies. We have shown that also in the ZmCPK11-transgenic plants the

activity of ZmCPK11 increases after wounding, touch and LA or MeJA treatment, whereas

the wound-induced ZmCPK11 activity was completely inhibited by acSA. The use of the

ZmCPK11-transgenic Arabidopsis plants and anti-c-Myc antibodies allowed us to confirm

that 56 kDa CDPK kinase is ZmCPK11, because ZmCPK11 kinase in transgenic plants showed

the same responses to wounding, touch and LA, MeJA or acSA treatment as an endogenous

enzyme in maize. The effect of LA, MeJA and acSA on the in vitro activity of recombinant

GST-ZmCPK11 was also investigated. Only LA stimulated in vitro kinase activity, suggesting

the possibility of direct interaction between LA and ZmCPK11.

Summing up: for the first time we showed the enzymatic activation of the CDPK/ZmCPK11

kinase as an element of the systemic response to mechanical wounding. In addition, we have

demonstrated that ZmCPK11 participates in JA-dependent local and systemic response to

wounding and in response to touch. The JA signaling pathway occurs earlier than ZmCPK11

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and its components, LA and MeJA, are positive regulators of expression and activity of

ZmCPK11.

CDPKs are involved in the regulation of response to wounding in dicots. AtCPK3 and

AtCPK13 positively regulate responses to wounding and herbivore (Spodoptera littoralis)

attack. They phosphorylate the HsfB2a transcription factor that is required for the

expression of PDF1.2 induced by a herbivore (Kanchiswamy et al. 2010). In contrast, silencing

in tobacco (Nicotiana attenuata) NaCDPK4 and NaCDPK5 resulted in an increase in JA

content after wounding or larvae (Manduca sexta) attack as well as an increase in defense

metabolites, suggesting that NaCDPK4 and NaCDPK5 are negative regulators of JA

biosynthesis and defense responses to wounding (Yang et al. 2012, Hettenhausen et al.

2013). The negative regulator of the cotton defense reactions (Gossypium hirsutum) for the

disease caused by the Verticilium dahliae fungus is GhCPK33. The GhCPK33 knockout mutant

has been shown to constitutively activate JA-dependent defense responses and enhance

immunity to Verticilium dahliae. GhCPK33 phosphorylates the enzyme of JA biosynthesis

pathway - 12-ketophytodienic acid reductase (GhOPR3). Phosphorylation of GhOPR3

destabilizes the enzyme, inhibits JA synthesis and, consequently lowers resistance to

Verticilium dahliae (Hu et al. 2018). In soybean (Glycine max), out of 39 CDPKs, only

expression of GmCPK3 and GmCPK31 increased after mechanical wounding or Spodoptera

litura caterpillar attack, which suggests the participation of these kinases in response to

wounding (Liu et al. 2016). Signal pathways in monocotyledonous plants in which CDPK

kinases participate are less known. We have isolated and cloned the HvCDPK12 cDNA, the

structural homologue of ZmCPK11. The HvCDPK12 cDNA sequence was deposited on the

GeneBank/EMBL database under the number EU240660.2. Like ZmCPK11, the activity and

level of HvCDPK12 transcript increased in barley leaves after wounding (activity after

minutes, transcript after 3-4 hours). In addition, HvCPK12 expression was increased in barley

leaves treated with JA or LA. The results obtained suggest that HvCDPK12 and ZmCPK11 are

orthologs in response to mechanical wounding. Results were presented at the conference

“Plant Abiotic Stress Tolerance”, 8-11 February 2009, Vienna, Austria (Lewandowska-

Gnatowska E, Szczegielniak J, Muszyńska G Wound-responsive Calcium dependent protein

kinase from barley - HvCDPK12) and are part of the PhD dissertation Elżbieta Lewandowska-

Gnatowska (planned defense by the end of this year). HvCPK12, as well as the closest

homologue of ZmCPK11 in rice, OsCPK24, shows 91% identity in the amino acid sequence of

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ZmCPK11. The level of the OsCPK24 transcript was shown to be very high in rice roots and

increases in response to cold, rice blast and chitin (Wan et al. 2007). In addition, ZmCPK11

has been shown to be involved in ABA-induced antioxidant defense by increasing the

expression and activity of antioxidant enzymes: superoxide dismutase (SOD) and ascorbic

peroxidase (APX) (Ding et al. 2013).

In many plant species, DNA methylation regulates gene expression in response to

biotic and abiotic stresses (Xu et al. 2018). Often, methylation of DNA in promoter

sequences is positively correlated with transcriptional gene silencing (Zilberman et al. 2007).

The analysis of the nucleotide sequence of the ZmCPK11 gene revealed the presence of DNA

segments rich in CpG dinucleotides called CpG islands in the promoter and first exon (gene

body) of ZmCPK11. This indicated the possibility of regulating the expression of ZmCPK11

through methylation/demethylation processes, because the methylation of cytosine to 5'-

methylcytosine (m5C) in CpG dinucleotides is one of the mechanisms regulating gene

expression (Suzuki and Bird 2008). Methylation also occurs in DNA sequence contexts CHG

and CHH (H = A, T, or C) and less, particularly in the regions of the DNA encoding the protein

(Gene Body Methylation, GBM).

Therefore, our next goal was to investigate the effect of wound stress on global

methylation of genomic DNA in maize as well as on methylation of the wound induced

ZmCPK11 gene. The obtained results are presented in the paper

Lewandowska-Gnatowska E, Polkowska-Kowalczyk L, Szczegielniak J, Barciszewska M, Barciszewski J, Muszyńska G. (2014) Is DNA methylation modulated by wounding-induced oxidative burst in maize? Plant Physiol Biochem 82: 202-208

Injury of maize leaves induces a reversible decrease, of the genomic DNA methylation level

by 20-30% within 1 h after wounding, which then return to the initial level within 2 h after

the stress. Removal of the methyl group from cytosine (demethylation) may proceed in an

active or passive manner. Glycosylases, DME (DEMETER) and ROS1 (Repressor of Silencing 1)

are involved in active demethylation (Xu et al. 2018), whereas passive demethylation can be

caused by reactive oxygen species, ROS) (Kreuz and Fischle 2016). It is also known that

mechanical wounding causes reactive oxygen species (ROS) “burst” and DNA damage (Apel

and Hirt 2004). Rapid and reversible changes in methylation of maize DNA in response to

wounding suggested the contribution of non-enzymatic reactions induced by ROS. We have

shown that wounding in maize leaves induces a two-stage increase in oxidative stress (ROS

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production), the first immediately after injury and the second two hours later. The kinetics of

ROS production in response to wounding differs significantly between species, ranging from

biphasic, for example in Medicago truncatula (Soares et al. 2009) to long-term and multi-

component in potato (Razem et al. 2003). These results indicated that the oxidative stress

induced by injury to maize leaves may contribute to transient changes in the level of

cytosine methylation in DNA. In addition, analysis of the methylation status of CpG islands in

ZmCPK11 revealed that wounding stimulates demethylation of cytosine 100 and 126 in the

first ZmCPK11 exon. Demethylation of these cytosines was correlated with the increase in

the level of the ZmCPK11 transcript after wounding, suggesting that the

methylation/demethylation processes can be involved in the regulation of the ZmCPK11

transcription in response to stress. The results obtained were consistent with the earlier

report that cytosines methylation in the coding region of the Phytochrome A gene silenced

its expression (Rangani et al. 2012). On the other hand, the analysis of global methylation

and transcription of the apple genome showed that the GBM of many genes was positively

correlated with their expression. While in many plants the function of DNA methylation in

the promoters is related to the silencing of gene transcription, the GBM function is very

poorly understood. GBM is very common in genes that are constitutionally transcribed,

highly conserved, and least frequently in the genes with the most variable expression

(Zilberman 2017).

Summing up: in this work we showed that demethylation of cytosine 100 and 126 in the

first ZmCPK11 exon is correlated with the increase in expression of ZmCPK11 in response to

mechanical injury, which may suggest that methylation/demethylation processes regulate

the expression of ZmCPK11 in response to stress. We have also shown that in maize, the

oxidative stress caused by injury is temporally correlated with transient DNA

hypomethylation.

We then studied the participation of ZmCPK11 in the response of the plant to other

stresses, including stress of salinity. An increase in enzymatic activity and expression of

ZmCPK11 in roots and leaves of maize in response to salt stress indicated that ZmCPK11 is

involved in the maize response to salinity. The results described below have been

published in the work

Borkiewicz L, Polkowska-Kowalczyk L, Cieśla J, Sowiński P, Jończyk M, Rymaszewski W,

Szymańska KP, Jaźwiec R, Muszyńska G, Szczegielniak J (2019) Expression of maize

14

Calcium Dependent Protein Kinase (ZmCPK11) improves salt tolerance in transgenic Arabidopsis plants by regulating sodium and potassium homeostasis and stabilizing photosystem II. Physiol Plant DOI: 10.1111/ppl.12938

For these studies we used the transgenic Arabidopsis plants producing c-Myc-ZmCPK11.

ZmCPK11 was activated in the roots and leaves of transgenic plants after 10 minutes from

the application of salt stress, suggesting the participation of ZmCPK11 in local and systemic

response to salinity in Arabidopsis. These results also suggest that ZmCPK11 may be part of a

documented signal system based on fast and long-distance calcium wave transfer in

response to salt stress (Choi et al. 2014). In order to understand the role of ZmCPK11 in plant

resistance to salinity, the phenotype of transgenic plants with c-Myc-ZmCPK11 was analyzed.

The influence of salt stress on seed germination, growth after germination and root growth

was investigated. No significant differences were observed in seed germination and in

growth after germination under optimal conditions for growth between control plants (wild

type, WT and transgenic plants with empty vector, eV4) and transgenic plants with c-Myc-

ZmCPK11. Phenotypic differences occurred under salt stress conditions. Transgenic plants

were more sensitive to salinity during germination of seeds than control plants, while during

growth after germination, as well as 4-week-old plants showed increased resistance to

salinity. Their leaves were less chlorotic and showed weaker symptoms of senescence.

Examination of chlorophyll content in the leaves, by spectrophotometric method, confirmed

our assumptions, since the lines of transgenic plants with c-Myc-ZmCPK11 under the

conditions of salt stress contained more chlorophyll a and b than control plants. Studies of

the maximum quantum yield of photosystem II (Fv/Fm), which is a parameter often used to

estimate the degree of damage to photosystem II and the emergence of photoinhibition

under stress conditions showed that transgenic plants with c-Myc-ZmCPK11 under salt stress

had, compared to the control plants, more efficient photosystem II.

In addition, we observed increased expression of genes encoding transcription factors (CBF1,

CBF2, CBF3, ZAT6 and ZAT10) and transporters of sodium (Na+) and potassium (K+) ions

(HKT1, NHX1 and SOS1) in the roots of ZmCPK11-transgenic plants in response to salt stress.

HKT1, SOS1 and NHX1 play an important role in plant tolerance for salinity by inhibiting the

transport of Na+ from roots to leaves, removing excess Na+ from roots into the environment

and their sequestration to vacuoles (Almeida et al. 2017). In contrast, CBFs and ZATs

regulate the transcription of genes encoding proteins involved in plant tolerance to stress.

15

Increased expression of genes encoding Na+ and K+ transporters in transgenic plants with c-

Myc-ZmCPK11 prompted us to check the content of these ions in the roots and leaves of the

tested plants. The results pointed to inhibition of Na+ transport from roots to leaves in the

ZmCPK11-transgenic plants under salt stress conditions. The leaves of these plants contained

less Na+ and had a higher K+/Na+ ratio, which is an indicator of the proper functioning of the

cell. The obtained results show that ZmCPK11 enhances salt tolerance of plants by increasing

the expression of genes related to maintaining homeostasis of Na+ and K+ ions and stabilizing

the photosynthetic system.

In addition, it has been shown that ZmCPK11 participates in primary root growth. Under

control conditions, it is a positive regulator of root growth, while under salt stress conditions

it is a positive regulator of root growth inhibition. ZmCPK11 is also participates in root

growth inhibition processes regulated by plant hormones: methyl jasmonate (MeJA) and

abscisic acid (ABA). The root growth of ZmCPK11-transgenic plants was more strongly

inhibited by MeJA and ABA compared to control plants (WT and eV4). This proves that

ZmCPK11 is a positive regulator of ABA- and MeJA-dependent root growth inhibition. The

study of the level of these hormones by liquid chromatography coupled with mass

spectrometry (HPLC-MS/MS) showed that the roots of transgenic plants expressing ZmCPK11

contained less tested phytohormones under control conditions than WT plants, while more

JA under salt stress. Considering that ABA and JA inhibit root growth in high concentrations,

the results obtained show that the phenotype of long roots of transgenic plants under

control conditions and short roots under salt stress conditions may be caused by changes in

level of endogenous phytohormons, ABA and JA.

Summing up: ZmCPK11 participates in the earliest stages of response to salt stress, in the

roots and leaves, of maize and Arabidopsis. ZmCPK11 increases the tolerance to salinity

through the following defense mechanisms:

- induces in the roots expression of genes encoding transcription factors (CBF1, CBF2, CBF3,

ZAT6 and ZAT10) and Na+ and K+ transporters (HKT1, NHX1 and SOS1), which play a role in

Increasing stress tolerance;

- regulates Na+ and K+ homeostasis by inhibiting the transport of toxic Na+ from roots to

leaves and maintains in leaves a high K+/Na+ ratio ensuring the proper function of the

cells;

- prevents the degradation of chlorophyll and stabilizes the photosynthetic system.

16

In addition, ZmCPK11 regulates root growth by regulating the level of ABA and JA.

Summary and (based on preliminary results) future perspectives

In conclusion, in my opinion, the most important research results in which I participated

was to demonstrate that:

1. ZmCPK11 participates in the earliest stages of local and systemic response to wounding

and salt stress in Zea mays and Arabidopsis thaliana;

2. ZmCPK11 participates in JA-dependent wound-signal transduction pathway and that the

components of this pathway (LA and MeJA) are positive regulators of activity and

expression of ZmCPK11. The kinase studied also participates in the touch response;

3. During wounding, the transcriptional and enzymatic activity of ZmCPK11 is regulated by

a secondary messenger - phosphatidic acid (PA), synthesized in the phospholipase D

pathway. Stimulation of ZmCPK11 activity by PA in response to wounding can be the result

of direct enzyme interaction with a phospholipid;

4. Demethylation of the two cytosines 100 and 126 in the first ZmCPK11 exon after injury is

correlated with the increase in the transcript level of this gene. This suggests that

methylation/demethylation processes under wound stress can regulate the expression of

ZmCPK11;

5. ZmCPK11 increases tolerance to salinity in Arabidopsis by regulating Na+ and K+

homeostasis, prevention of chlorophyll degradation and stabilization of the

photosynthetic system; ZmCPK11 increases the expression of genes encoding Na+ and K+

transporters (HKT1, NHX1 and SOS1) and transcription factors (CBF1, CBF2, CBF3, ZAT6

and ZAT10), genes involved in plant tolerance processes for stress;

6. ZmCPK11 regulates root growth:

- under control conditions it is a positive regulator of root growth;

- under the conditions of salt stress (NaCl) is a positive regulator of root growth inhibition;

- is a positive regulator of root growth inhibition in ABA and JA-dependent pathways;

- ZmCPK11 regulates root growth by regulation of ABA and JA levels.

We plan to continue studying the role of ZmCPK11 in abiotic stress. We will focus on

the research of two potential substrates ZmCPK11. One of them is aldehyde dehydrogenase

17

(ZmRF2). Its closest homologue in Arabidopsis, ALDH2B4 (74% identity in the amino acid

sequence of ZmRF2), was identified as a protein that co-immunoprecipitated with c-Myc-

ZmCPK11 from the extract of injured leaves of the ZmCPK11-transgenic Arabidopsis plants.

The second potential substrate is the transcription factor AtZAT6, the expression of which

increases significantly in response to salt stress in transgenic Arabidopsis plants expressing

ZmCPK11. The results of our studies show that ZmRF2 and AtZAT6 are phosphorylated in

vitro by recombinant GST-ZmCPK11. We got to know three phosphorylation sites in ZmRF2

by mass spectrometry analysis and demonstrated the interaction of ZmRF2 with ZmCPK11 in

vivo by the BIFC (Bimolecular Fluorescence Complementation) method and using a transient

expression system in protoplasts from Arabidopsis. We plan to examine whether the closest

homologs of ZmCPK11 in Arabidopsis, AtCPK4 and AtCPK11, phosphorylate AtZAT6 and

ALDH2B4 in vitro and to identify the phosphorylation sites. We are also planning to learn

about the role of these phosphorylations.

In addition, we plan to check whether AtCPK4 and AtCPK11 play a similar role to

ZmCPK11 in responses to wounding and salinity. We will use the ZmCPK11- transgenic

Arabidopsis plants and knockout mutants in the AtCPK4 and AtCPK11 genes for research.

The results of our research show that ZmCPK11 stimulates the expression of JA biosynthesis

and signaling genes (AOC2, AOS2, VSP2, ORA59, TAT3) in response to mechanical wounding,

but inhibits the expression of JA-dependent genes, repressor JAZ1. According to our

preliminary results, AtCPK4 and AtCPK11 also act as positive regulators of genes involved in

JA biosynthesis and signaling in response to wounding, because the transcript level of these

genes in single (cpk4 and cpk11) and double cpk4/cpk11 mutants was significantly reduced

compared to wild type plants (results were shown at the 27th International Conference on

Arabidopsis Research conference, June 29 - July 3, 2016, Gyeongju, Korea). Research on the

role of ZmCPK11 and its homologs, AtCPK4 and AtCPK11, in abiotic stresses will be

continued.

5. Discussion of the other scientific achievements.

In 1983, I started working at the Department of Plant Biochemistry at the Institute of

Biochemistry and Biophysics of the Polish Academy of Sciences in a team headed by prof.

Danuta Wasilewska. The result of research in which I participated was a publication

18

Wasilewska et al. (1987). In 1986, I joined the team of prof. Grażyna Muszyńska at IBB PAS,

but due to my son's health problems, I worked part-time for the first three years. At that

time, the team of prof. Grażyna Muszyńska started to work with the processes of reversible

phosphorylation of proteins in plants, and in particular with enzymes regulating these

processes: kinases and protein phosphatases. I joined the research on calcium (Ca2+)-

dependent and phospholipids stimulated protein kinase, which I am working on to this day.

Research on this kinase was carried out in cooperation with the Laboratory headed by prof.

Lorentz Engström at the Department of Medical and Physiological Chemistry, Biomedical

Center in Uppsala (Sweden), who for many years worked on animal protein kinases,

including protein kinase C (PKC). My several-week three stays in the laboratory in Uppsala

contributed to the creation of two publications whose results were included in my doctoral

dissertation (Lindblom et al. 1997, Szczegielniak et al. 2000). In the paper

Lindblom S, Ek P, Muszyńska G, Ek B, Szczegielniak J, Engström L (1997) Phosphorylation of sucrose synthase from maize seedlings. Acta Biochim Polon 44: 809-818

we were looking for endogenous substrates of Ca2+-dependent and phospholipids stimulated

protein kinase from maize seedlings. For this purpose, phosphorylation of co-purifying

proteins with the studied kinase by endogenous protein kinase was performed using [γ-32P]

ATP as a phosphate donor. Among the most heavily phosphorylated proteins, we have

focused on the study of protein with molecular weight of 86 kDa, and therefore

phosphorylated 86 kDa protein was resolved by SDS-PAGE, subjected to enzymatic

proteolysis and the amino acid sequence of the 10 isolated peptides was determined. The

sequences of these peptides showed complete homology to two sucrose synthase

isoenzymes (SS1 and SS2). This was the first information regarding the identification of

sucrose synthase as an endogenous substrate of Ca2+ dependent and phospholipids

stimulated protein kinase. Sucrose synthase catalyzes the sucrose degradation reaction. Two

isoenzymes of sucrose synthase (SS1 and SS2) encoded by two genes (Sh1 and Sus1) were

discovered in maize (Echt and Chourey 1985). The next stage of our research was the

identification of phosphorylated amino acid residues in sucrose synthase by endogenous

kinase. The obtained results showed that serine-15 only in the sucrose synthase SS2

isoenzyme is phosphorylated in vivo by the studied kinase. in vitro studies on a synthetic

peptide containing sequences surrounding serine-15 in SS2 confirmed the observation that

the serine located at the N-terminus of the SS2 isoenzyme is phosphorylated by Ca2+-

19

dependent and phospholipids stimulated protein kinase. These results were in line with

previous observations that calcium-dependent phosphorylation activates sucrose synthase

from maize leaves and that the phosphorylated amino acid is serine located at the N-

terminus of sucrose synthase (Huber et al. 1996).

Previously, the activity of Ca2+-dependent and phospholipid stimulated protein kinase

resembling animal PKC has been identified in maize seedlings (Muszyńska et al. 1993). The

aim of these studies was further detailed characterization of this protein kinase. Therefore,

we attempted to purify this kinase, followed by the biochemical and immunological

characterization of the purified enzyme. The study results were described in the publication

Szczegielniak J, Liwosz A, Jurkowski I, Loog M, Dobrowolska G, Ek P, Harmon AC, Muszyńska G (2000) Calcium-dependent protein kinase from maize seedlings activated by phospholipids. Eur J Biochem 267: 3818-3827

The protein kinase studied was isolated from etiolated maize seedlings and partially purified

by hydrophobic chromatography (Octyl-Sepharose), anion exchange chromatography (DEAE-

52, followed by Mono Q) and affinity chromatography (MBP-Sepharose). The purified kinase,

with a 54 kDa molecular weight determined by gel chromatography, was named ZmCPKp54.

Kinase activity was stimulated by lipids such as PS, PI, arachidonic acid and endogenous

lipids isolated from maize seedlings. Activation by PS ZmCPKp54 resembled animal PKC, but

the plant enzyme was different from typical PKCs due to the lack of sensitivity to

diacylglycerol and phorbol esters. Immunological studies showed that ZmCPKp54 was

recognized by antibodies directed against a calmodulin-like CDPK domain from soybean, but

not by antibodies directed against the catalytic or regulatory PKC domain. The maize enzyme

phosphorylated exogenous substrates phosphorylated by both CDPK and PKC: histone H1

and two synthetic peptides derived from glycogen synthase. However, the plant enzyme did

not phosphorylate two synthetic peptides of specific PKC substrates (one derived from the

PKC pseudosubstrate sequence and the other derived from the MARCKS protein). Two

specific PKC inhibitors: GF 109203X bisindolylmaleimide and a synthetic peptide derived

from the PKC pseudosubstrate sequence did not affect both the maize protein kinase activity

and soybean CDPK activity. The substrate specificity and sensitivity to the inhibitors of the

maize enzyme resembled CDPK. The presented research results have provided strong

evidence that the protein kinase studied is one of the CDPK isoforms and probably a

functional PKC analog that can participate in signal transduction pathways. In these studies,

20

we received invaluable help from prof. Alice Harmon (University of Florida, Gainesville,

Florida, ACH), who was one of the first scientists to work on CDPK.

For several years, I started to cooperate with dr Lidia Polkowska-Kowalczyk (IBB PAS) in

potato CDPK research and the results of this collaboration is publication

Gromadka R, Cieśla J, Olszak K, Szczegielniak J, Muszyńska G, Polkowska-Kowalczyk L (2018) Genome-wide analysis and expression profiling of calcium dependent protein kinases in potato (Solanum tuberosum). Plant Growth Regul 84: 303-315

The main goal of the research was to identify the CDPK genes in potato (Solanum

tuberosum) and to analyze their expression in various organs. The availability of the entire

potato genome sequence (Potato Genome Sequencing Consortium 2011) made it possible to

identify in silico 23 CDPK genes. According to the predicted amino acid sequences, the

protein kinases identified have a typical CDPK structure. It was also identified eight CDPK-

related kinases (CRKs) with less than four "EF-hand" motifs in the calmodulin-like domain.

CRKs are also found in the known genomes of other plants. Of the 23 known CDPKs in

potato, 14 at the N-terminus contain the predicted myristylation site, which plays an

important role in protein interactions with membranes and other proteins. Potato genome

analysis showed that CDPK genes are found on 11 chromosomes, of 12, potato

chromosomes. Based on phylogenetic analysis and gene structure, CDPKs from potato were

divided into four subgroups similarly to known CDPKs from other plants. Expression analysis

by RT-qPCR revealed that the CDPKs were expressed in all the organs, but their expression

patterns varied greatly. This suggests that CDPKs can play specific roles in potato

development. In addition, changes in the activity and expression profiles of CDPKs were

investigated in three Solanum genotypes with different types and level of resistance against

Phytophthora Infestans. Leaves of Solanum species were treated with elicitor (culture filtrate

of P. infestans, CF). In our experimental conditions, the magnitude and duration of gene

induction and increase of CDPK activity correlated positively with the level of resistance of

the Solanum genotypes to P. infestans. Increased CDPK expression and activity in response

to the elicitor from P. infestans indicates involvement of CDPKs in the defence against this

pathogen.

I also participated in the study of casein kinases 2 (CK2). CK2 are constitutively active,

highly conserved protein kinases in all eukaryotic organisms. CK2 is a heterotetramer

composed of 2 catalytic subunits or ' (CK2 ) and 2 regulatory subunits (CK2 ). CK2

21

has kinase activity, whereas CK2 is a regulatory subunit that stimulates CK2 activity and

stabilizes the heterotetramer. Over 300 endogenous CK2 substrates are known in animals

(Litchfield 2003, Meggio and Pinna 2003). These proteins perform important biological

functions in the processes of proliferation, differentiation, transformation, inhibition of

apoptosis, transcriptional control and others (Litchfield 2003, Bibby and Litchfield 2005). I

joined the research conducted by Grażyna Dobrowolska, PhD student Prof. Grażyna

Muszyńska. Research on CK2 was carried out in cooperation with a laboratory led by prof.

Lorenzo A. Pinna in the Department of Biological Chemistry at the University of Padua (Italy),

who was a specialist in animal casein kinases. At this stage of the research I participated in

the purification and characterization processes of both casein kinases and protein

phosphatases from maize seedlings, and the obtained results were published in three

publications.

A typical oligomeric form of casein kinase with a molecular weight of about 135 kDa,

named CKIIA, and a monomer form with a molecular weight of about 39 kDa, named CKIIB,

was identified in maize seedlings (Dobrowolska et al. 1987). This was the first report on the

possible existence of kinases in plants with the features of the free catalytic CK2 subunit.

Therefore, at work

Dobrowolska G, Meggio F, Szczegielniak J, Muszyńska G, Pinna LA (1992) Purification and characterization of maize seedlings casein kinase IIB, a monomeric enzyme

immunologically related to the subunit of animal casein kinase-2. Eur J Biochem 204: 299-303

CKIIB was purified to homogeneity and biochemical and immunological characteristics of

the enzyme were performed. In terms of substrate specificity, CKIIB was similar to the CK2α.

CKIIB phosphorylated animal CK2 substrates together with the human CK2 , which is

phosphorylated by animal CK2α. In immunological terms, CKII also resembled CK2α because

it responded to antibodies directed against animal CK2α, while it did not respond to

antibodies directed against animal CK2β. Anti-CK2α antibodies recognized 39 kDa protein in

both CKIIB and CKIIA preparations. The results of these studies indicated that CKIIB from

maize seedlings represents a naturally occurring monomeric form of the kinase, without a

regulatory subunit, which was later confirmed by other researchers (Espunya and Carmen

1997).

22

The presence of high activity of casein and histone phosphorylating kinases in extracts

from maize seedlings led us to study the reverse processes for phosphorylation –

dephosphorylation. Therefore, the next publication which was created in cooperation with

prof. Lorenzo Pinna concerned protein phosphatases. The aim of the study was to identify

and characterize phosphatases from maize seedlings and their comparison with typical

animal protein phosphatases. Part of this research was done during my monthly stay in the

laboratory of prof. Lorenco Pinna. The obtained results were described in the paper

Jagiełło I, Donnela Deana A, Szczegielniak J, Pinna L A, Muszyńska G (1992) Identification of protein phosphatase activities in maize seedlings. Biochim Bioph Acta 1134: 129-136

We were isolated from maize seedlings and partially purified by ion-exchange

chromatography (DEAE-cellulose), affinity chromatography (Heparin-Sepharose) and gel

chromatography, three enzyme fractions with phosphatase activity named PCP-I, PCP-II and

PCP-III (PhosphoCasein Phosphatases). The results of the biochemical analysis of the enzyme

fractions showed that PCP-I and PCP-II do not have the characteristic features of protein

phosphatases, type 1 (PP1) and type 2A (PP2A), but their substrate specificity, insensitivity to

protein phosphatase inhibitor - okadaic acid and binding to the bed with immobilized

heparin showed that they are acid phosphatases. PCP-III, on the other hand, was identified

as the catalytic subunit of protein phosphatase 2A (PP2A). The following findings made it

possible: (1) The molecular weight of PCP-III was consistent with the molecular weight of

PP2A; (2) PCP-III showed a substrate specificity similar to PP2A; (3) PCP-III did not bind to the

bed with immobilized heparin, which is a characteristic of PP2A but not PP1; (4) PCP-III

activity was inhibited by similar concentrations of okadaic acid as PP2A activity. In addition,

in this work, a new known feature of acid phosphatases was their binding to heparin

immobilized on the bed. This feature could be used in the future both for the purification of

acid phosphatases and for the separation of acid phosphatase activities from the activity of

protein phosphatases.

After many years, inspired by prof. Grażyna Dobrowolska, I returned to the study of

casein kinases and published the results of our studies at work

Łebska M, Szczegielniak J, Cozza G, Moro S, Dobrowolska G, Muszyńska G (2009) A novel splicing variant encoding putative catalytic α subunit of maize protein Kinase CK2. Physiol Plant 136: 251-253

23

Previously, three cDNA clones encoding the catalytic subunit α (CK2α) were isolated and

characterized in maize (ZmCK2α-1, ZmCK2α-2 and ZmCK2α-3) (Dobrowolska et al 1991,

Peracchia et al. 1999, Riera et al. 2001). In these studies, we undertook cloning of the new

CK2α isoforms. From the cDNA library of 5-week-old maize leaves I isolated the cDNA of the

fourth, new CK2α, named ZmCK2α-4. The ZmCK2 -4 sequence has been deposited in the

GeneBank nucleotide database under number AAF76187. The predicted amino acid

sequence of ZmCK2 -4 showed the highest homology with ZmCK2α-1. According to the

analysis of all maize CK2α sequences, ZmCK2α-4 and the previously identified ZmCK2α-1

(accession no. X61387) differ only in the first exon and are most likely alternatively

transcribed from the same ZmPKCK2AL gene (accession No. Y11649, Peracchia et al. 1999).

In addition, the N-terminal fragment of ZmCK2α-4 was different from the N-terminus of all

CK2α known to date from various organisms. The next stage of the research was to check if

the new, cloned form CK2 is a functional protein. Because of the three potential translation

start sites in ZmCK2α-4 mRNA we produced in the bacterial system three variants of

ZmCK2α-4 as fusion proteins with GST and checked their activity. In contrast to the known

CK2αs, all three variants of GST: ZmCK2α-4 and ZmCK2α-4 (variant III, no protein tag) were

catalytically inactive, both as monomeric proteins as well as heterotetramers after the

addition of CK2β. We were surprised by this result and therefore we undertook to explain

the cause of the lack of enzymatic activity of the newly known CK2α. The cause of the

observed inactivity was in the first exon, because it was the only difference between inactive

ZmCK2α-4 and active ZmCK2α-1. Based on the previously known crystalline structure of

ZmCK2α-1 (Niefind et al. 1998), we created the structural model ZmCK2α-4 in cooperation

with Dr. Giorgio Cozza and Dr. Stefano Moro from the University of Padua (Italy). The

developed model showed a lack in ZmCK2α-4 of several important interactions present in

ZmCK2α-1. Instead of Ala4, Arg5, Tyr7 and Tyr21 present in ZmCK2α-1, in ZmCK2α-4 there

are Leu7, Cys8, Trp10 and Thr24. These changes cause that the N-terminus of ZmCK2α-4 is

unable to interact with the activation loop and maintain it in an open and full active

conformation. These observations were in agreement with the results of Sarno et al. (2002)

showing that the CK2α deletion mutants in the N-terminal segment almost lost their

catalytic activity. The study of the expression in various organs at different stages of

development detected the ZmCK2α-4 transcript in 8-week leaves and roots, but not in 4-day-

old seedlings, roots and 2-week-old leaves. In contrast to ZmCK2α-4, the ZmCK2α-1

24

transcript was present in all organs tested, younger and older. Considering the fact that CK2

is a positive regulator of proliferation, it is possible that in older organs, when plant growth

is slowed down, ZmCK2α-4 counteracts the expression of ZmCK2α-1. Our preliminary results

also show that ZmCK2α-4 can function as a negative regulator of phosphorylation of certain

proteins phosphorylated by endogenous CK2 and as a holoenzyme whose catalytic activity is

stimulated by a specific regulatory subunit(s).

A few years later, a new form of CK2α, named CK2α4 (GRMZM2G141903) was identified in

maize and supplemented to a full-length form CK2α2 (GRMZM2G047855) (Vélez-Bermúdez

et al. 2015). The CK2α2 and CK2α4 proteins are composed of 383 amino acids and are larger

than the cloned ZmCK2α-4 (368 amino acids). The N-terminal domains of CK2α2 and CK2α4

contain the cTP signal peptide that targets the protein to chloroplasts. The authors of the

study showed that the cTP peptide is responsible for the location of both kinases and

binding to the CK2 regulatory subunit CK2β. CK2α4 was localized in chloroplasts, while

CK2α2 in addition to chloroplasts, in the cytoplasm and the endoplasmic reticulum. The

chloroplast location of CK2α2 and CK2α4 suggests that they can play important functions in

the photosynthesis process.

We continued research on CK2 function in maize by searching for endogenous

substrates of these kinases. The results obtained were described in the next publication

Łebska M, Ciesielski A, Szymona L, Godecka L, Lewandowska-Gnatowska E, Szczegielniak J, Muszyńska G (2010) Phosphorylation of maize eIF5a by CK2: identification of phosphorylated residue and influence on intracellular localization of eIF5a. J Biol Chem 285: 6217-6226

Isolated and purified kinase fractions, based on the previously developed method

(Dobrowolska et al. 1992) were separated by SDS-PAGE and the gel fragments containing

kinases analyzed by mass spectrometry. Of the identified co-purifying proteins with the

fraction containing the free catalytic subunit CK2 (CK2 ), the eukaryotic translation

initiation factor 5A (eIF5A) was chosen for further characterization as a potential CK2

substrate. eIF5A was cloned and the recombinant protein was produced in a bacterial

system. We have demonstrated that eIF5A is phosphorylated in vitro by endogenous CK2

as well as by the recombinant ZmCK2 -1 enzyme. We also confirmed the phosphorylation in

vivo of eIF5A by the separation of maize seedlings proteins by means of two-directional

electrophoresis and the identification of separated phosphoproteins by means of mass

25

spectrometry. The analysis of the amino acid sequence of eIF5A from different plants

showed the existence in eIF5A of monocotyledonous plants of two potential

phosphorylation sites by CK2, serine 2 and serine 4. In animals and dicotyledonous plants,

there is no serine 4 in eIF5A. To identify which serine residues (Ser-2 and/or Ser-4) are

phosphorylated in ZmeIF5A, we mutated the serine residue to alanine, which is not

phosphorylated. We then produced in the bacterial system wild-type ZmeIF5A and its

mutant variants, ZmIF5A-Ser2Ala and ZmIF5A-Ser4Ala, and checked whether they were

phosphorylated by CK2. Of the recombinant proteins, only ZmIF5A-Ser2Ala was not

phosphorylated by CK2 from maize. eIF5A from Arabidopsis and yeast (Saccharomyces

cerevisiae) was also efficiently phosphorylated by CK2, but their mutant forms (Ser2Ala) did

not attach phosphate groups. The results obtained indicated that Ser-2 is phosphorylated by

CK2. To confirm the phosphorylation sites, these phosphoproteins were analyzed by mass

spectrometry. However, we have not been able to detect any phosphorylation, most likely

due to localization in the end of protein. The new method we developed (described in the

paper), using specific proteolytic activity of thrombin, confirmed that Ser-2 in ZmeIF5A is

actually phosphorylated by CK2. This is a very interesting method that can be used to

confirm the postulated phosphorylation sites in other proteins. The use of confocal

microscopy and the transient expression system in protoplasts from mesophyll of maize

leaves made it possible to study the role of Ser-2 phosphorylation in ZmeIF5A. For this

purpose, we have prepared appropriate gene constructs containing the wild type ZmeIF5A

and mutants. In one mutant, the serine was converted to neutral alanine, whereas in

another mutant the serine was converted to an acidic amino acid, aspartic acid, which

mimics phosphoserine. The transient expression of the mentioned constructs in maize leaf

protoplasts showed that conversion of Ser-2 to aspartic acid in ZmeIF5A causes a three-fold

greater accumulation in the nucleus of the ZmeIF5A-Ser2Asp mutant. These results show

that reversible Ser-2 phosphorylation in ZmeIF5A may play a role in the regulation of eIF5A

transport between the nucleus and the cytoplasm. In this study, eIF5A was first recognized

as an endogenous CK2 substrate.

Currently, approximately 50 CK2 substrates are known in plants (Vilela et al. 2015a). Among

them, for example, ZmOST1 kinase, also known as SnRK2.6 or SnRK2E. CK2 phosphorylates

ZmOST1 and affects its activity, protein levels and stress responses. The ZmOST1-

Arabidopsis transgenic plants are more resistant to drought and hypersensitive to ABA with

26

respect to stomatal cells (Vilela et al. 2015b). In addition, transcription factors are CK2

substrates. Some of them, such as EmBP-2 and ZmBZ-1 in maize, associated with ABA-

dependent responses, regulate the expression of rab28 through ABRE elements.

Phosphorylation of EmBP-2 by CK2 strongly enhances its binding to DNA, whereas

phosphorylation of ZmBZ-1 weakens binding to DNA (Nieva et al. 2005).

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