Accardi L., Freudenberg W., Obya M. (Eds.) Quantum Bio-informatics IV: From Quantum Information to Bio-informatics

430

(A)

p-~

~

-

~.nl."""n.LS

3C

.'

·

Apoaequorin

Aequorin

(B)

Stimulation

-"

I

-

~

Plant cultured cells

Realtime monitoring

by luminometer

Figure

3. A non-invasive monitoring system for intracellular Ca

2

+ concentration in plant cells by

expressing a Ca

2

+ sensitive chemiluminescent protein, aequorin. (A) Aequorin

is

composed

of

an

apoprotein, apoaequorin, with

an

approximate molecular mass

of

22 kDa, and a chromophore,

coelenterazine. Aequorin has three EF-hand motifs that function

as

binding sites for Ca

2

+.

When

Ca

2

+ occupies these sites, the protein undergoes a conformational change and converts

coelenterazine into excited coelenteramide through oxidation, followed by emission

of

blue light

through relaxation to the ground state. (B) A realtime noninvasive Ca

2

+ monitoring system for

cultured plant cells by using a luminometer.

In tobacco cultured cells BY-2, cell cycle-depending synchronous defense

responses, including programmed cell death

(PCD), are induced

in

response to

cryptogein, a proteinaceous elicitor derived from a pathogenic oomycete,

Phytophthora cryptogea. Immediately after recognition

of

cryptogein, transient

and biphasic changes in [Ca

2

+]cyt

are induced (Fig.

4A)7,

subsequently followed

by

ROS production. Pharmacological analyses indicated that the two phases

of

the [Ca

2

+]cyt changes correspond

to

Ca

2

+ influx through the plasma membrane

and an inositol 1,4,5-trisphophate-mediated release

of

Ca

2

+ from intracellular

Ca

2

+ stores, respectively7. Chitin oligosaccharides and sphingolipid MAMPs

induced Ca

2

+ transients mainly due

to

the plasma membrane Ca

2

+ influx in rice

cells

8

(Fig. 4B). Considering that various patterns

of

changes in [Ca

2

+]cyt are

dependent on trigger molecules from pathogens, Ca

2

+ influx in defense

responses may be controlled by a series

of

regulatory mechanisms composed

of

Ca

2

+ influx through the plasma membrane and release

of

Ca

2

+ from intracellular

Ca

2

+ stores.

431

(A)

_1st

peak

(8)

_1st

peak

[Ca

2

'1

CY!

[Ca

z'

l

CY!

:;-

::s

~

c

Q)

.§

~

.~

~

o Q)

GlcNAc

5-

.5

cryptogein

fr

.5

~~

~

2nd

peak

Q) E

«..2

·EO

Q)

.<::

u

-5

0

5

10 15

-5

0

5

10

15

Time (min)

Figure

4. Two typical temporal patterns

of

changes in cytosolic Ca

2

+ concentraion triggered by

signal molecules from pathogens. Transformed tobacco and rice cells stably expressing aequorin

were used. (A) Cryptogein-induced changes in

[Ca

2

+],y"

in

tobacco BY-2 cells. (B) N-

acetylchitooligosaccharides-induced changes

in

[Ca

2

+]

,y

" in rice cells. rlu: relative luminescence

unit.

4.

Ca

2

+-permeable cation channels and plant immunityl-3

Activated Ca

2

+-permeable channels localized in the plasma membrane and

endomembranes such as vacuolar membrane mobilize Ca

2

+ into the cytosol

in

an

electrochemical potential-dependent manner. Consequently, spatio-temporal

patterns

of

[Ca

2

+]cyt

are formed. Little

is

known about the molecular bases for

the Ca

2

+ channels

l

-

3

•

In the early stage

of

defense responses, membrane

potential changes

9

and Ca

2

+ influx 7,8 occur at the same time, suggesting that

voltage-dependent Ca

2

+ channels (VDCCs) may be involved in these responses.

Vertebrate VDCCs are found in excitable cells

of

skeletal muscle, cardiac tissue,

and neurons. No homologues

ofVDCCs

have been found in plants. In contrast,

homologues

of

mammalian two-pore channels (TPCs) have been identified in

several plant species, such

as

Arabidopsis, rice, and tobacco

ls

-

l8

. Plant TPC

family proteins have two EF-hand motifs, putative Ca

2

+ binding domains, in the

intracellular domain, implying that the protein could

be

under feedback

regulation by binding

of

Ca

2

+. AtTPCI from Arabidopsis has been reported

to

encode a vacuolar membrane-localized cation channel and be involved in

sucrose-induced Ca

2

+ influx and stomatal Ca

2

+ response

lS

,

l6.

In contrast, rice

and wheat

TPCI are thought

to

localize

in

the plasma membrane

17

,19. Reverse

genetics studies suggest that

OsTPCI from rice

14

and NtTPCIA, B from

tobacco

18

are involved

in

plant immunity at least in suspension-cultured cells.

432

5. Regulation

of

Ca

2

+ -permeable channels

2

,3

How are Ca

2

+-permeable channels activated or inactivated in plant defense

responses? Oligosaccharide and sphingolipid MAMPs-induced Ca

2

+ transients

were dramatically suppressed by a protein phosphatase lI2A inhibitor, calyculin

A (CA) in

riceS.

When transient [Ca

2

+]cyt

changes are induced, Ca

2

+ channels

need

to

be negatively regulated immediately after they are activated. These

results suggest that protein phosphorylaion could be involved in the

downregulation

of

Ca

2

+ channels after transducing the signals in plant innate

immunity. However, MAMP-activated plasma membrane Ca

2

+-permeable

channel has not been identified, and molecular mechanisms for CA-induced

negative regulation

of

Ca

2

+ rise remain to be elucidated. Searches for the

phosphoproteins involved in the regulation

of

Ca

2

+ mobilization are currently

underway

to

further understand the relationship between Ca

2

+ signaling and the

regulation

of

plant innate immunity.

If

••

OFF Ca

2

;.

(Inactive)

ON

(Active)

Figure

5.

Reversible binding

of

Ca

2

+ to sensor proteins play key roles as a molecular switch.

6.

Decoding

of

Ca2+

-mediated signals by

Ca2+

sensor proteins

1

,20.22

How are the signals mediated by spatio-temporal patterns

of

[Ca

2

+]cyt

transmitted

to

downstream events? Many Ca

2

+ binding proteins containing an EF-hand

motifs

23

,24, including calmodulin (CaM), calcineurin B-like protein (CBL), and

Ca

2

+-dependent protein kinase (CDPK), have been identified in plants (Figs. 5

and 6), and play pivotal roles as sensors for Ca

2

+.

These proteins bind

to

ci

+

and change their conformation or three-dimensional structure to control the

activity or function

of

target proteins

as

"molecular switches". Ca

2

+/Ca

M-

dependent kinase (CCaMK)

is

has also been shown to be involved in decoding

the specific-pattern

of

[Ca

2

+]cyt

changes (Ca

2

+-spiking) in response

to

Nod

factors, signal molecules from symbiotic bacteria, during nodulation in legumes.

III

e

·

. "

..

....

c

CaM

1.

CaM

II

~

2.

CBL-CIPK

I

WUUW

1111

I

-11"-

"

[£OJ

. -

tJ

-

3.

CDPK

•

WUUW

•

....

1

Ca

2

+

kinase domain 0

EF

hand

II!I

CaM binding

FISL motif

(CBl

binding domain)

433

Figure

6.

The structure

of

representative Ca

2

+ sensor proteins

in

plants. Many genes for Ca

2

+

binding proteins containing Ca

2

+-binding EF-hand motifs, including calmodulins (CaM), calcineurin

B-like proteins

(CBL), and Ca

2

+-dependent protein kinases (CDPK), have been identified. These

proteins bind

to

Ca

2

+ and change their conformation

to

control the activity or function

of

target

proteins; therefore, each plays the role for

"Ca

2

+ sensor".

I Active

01.

~

.,<

-

Protein "

phosphatase

\0

®

Signal ON Signal OFF

•

Ca2+

RIIII!III kinase domain

II!lI

eBl

binding domain

Figure

7.

Molecular mechanisms for decoding

of

Ca

2

+-mediated signals by the Ca

2

+ sensor protein,

CBL and the protein kinase, CIPK. Note that both CBL and CIPK act as reversible molecular

switches.

434

The mammalian calcineurin consists

of

a type 2B protein phosphatase

(subunit A) and a Ca

2

+ sensor (subunit B). Many genes encoding calcineurin B-

like proteins (CBL), highly homologous to calcineurin

B,

exist in the plant

genome. However, neither a homologues

of

calcineurin A nor type

2B

protein

phosphatases has been found in plants. The CBLs bind to and activate a group

of

protein kinases, CBL-interacting protein kinases (CIPKs), in a Ca

2

+_

dependent manner (Fig.

7).

The model plant Arabidopsis thaliana has

10

CBLs

and

25

CIPKs. Their combinations for intermolecular interaction are

presumably involved in various Ca

2

+ -mediated signal transduction pathways.

Various CBL-CIPK systems play important roles in signal transduction

pathways triggered by abiotic stresses, such as wounding, low temperature,

drought, and

salro-

22

.

We recently identified CIPK proteins (OsCIPK14115) that

are induced by multiple MAMPs and function in innate immunity in rice.

Functional characterization

of

the OsCIPK14115-suppresed lines established by

RNAi method,

as

well

as

the overexpressing lines, suggested that these CIPKs are

involved in the regulation

of

various immune responses, including mitochondrial

dysfunction, PCD, biosynthesis

of

phytoalexins and pathogenesis-related gene

expression

as

well

as

ROS generation triggered by a protein from a fungal

pathogen

I2

,25.

The protein kinase activities

of

these CIPKs were enhanced by

combination with a specific CBL, OsCBL4.

Some

OsCBLs, including OsCBL4,

or other unidentified

factors

that interact with OsCIPK15 via the FISLINAF-motif

may regulate the activity and localization

of

OsCIPK14115 in the MAMP-

triggered signal transduction

pathwa/

2

.

Searches for the

in

vivo substrates

of

these CIPKs are currently underway to further elucidate the Ca

2

+ signaling

pathways regulating hypersensitive cell death and innate immunity. These

findings should shed further light on our understanding

of

defense signaling

pathways (Fig. 8).

Pa

thog

enic

sIgna

l

ca

;

(PAMPIMAMP)

Recep:.

~

.......

..

..........

~

'\1

Ca"channel

,

...

0'

, Spa

liot

e

mpor

al

cha

ng

es

of

[Ca

2

+1cyt

Ca

2

+ I

Intrace

llu

lar

Ca2+

pools Ca

2

+se

n

sors

Figure

8.

A hypothetical model for Ca

2

+-mediated immune responses in plants.

435

7. Concluding remarks

The Ca

2

+-mediated signal transmission system composed

of

the spatio-temporal

pattern formation

of

the intracellular Ca

2

+ concentration by Ca

2

+-permeable

channel proteins in the membranes, and its decoding by Ca

2

+ sensor proteins

is

a

universal information-processing system common to animals and plants.

However, molecules involved and the control mechanisms show marked

diversity. Ca

2

+ also plays a pivotal role

as

an information-processing element in

plant immunity. Identification

of

the molecules involved in these intracellular

signaling networks should be a key step to understand the mechanisms for

information processing in plants that do not evolve brains.

The understanding and utilization

of

the latent potentials

of

plants and

effectively coexisting with them are essential tasks for humans in the future.

Attempts to reduce the use

of

chemical pesticides including bactericidal agents

and insecticides by increasing plant immune responses have recently been

attracting attention. The genetic strengthening

of

disease resistance by breeding

and genetic engineering as well

as

exploration and the use

of

physiologically

active substances to enhance plant immune responses should markedly alter

next-generation agriculture. We recently discovered novel chemical substances

that potentially activate plant immune responses. Elucidation

of

the action

mechanism and evaluation for practical application are underway. The basis

of

these studies

is

understanding

of

the uniquely developed immune system

of

plants at a molecular level, which

is

completely different from that

of

animals.

Understanding the signal transmission network in plant immunity

is

an

important research subject in many contexts. Collaborative study between

molecular biologists and information scientists should be a challenging but

promising field in the near future.

Acknowledgement

This work was supported in part by Grant-in-Aid for Scientific Research on

Innovative Areas

(21200067) to T.K., for Exploratory Research (21658118)

to

K.K. and for Young Scientists (B) (21780041)

to

T.K., and by grants from Japan

Science and Technology Agency, for Adaptable and Seamless Technology

transfer

Program through target-driven R&D (AS221Z03504E) to T.K, and by a

grant from the Ministry

of

Agriculture, Forestry and Fisheries

of

Japan

(Integrated research project for plants, insects, and animals using genome

technology

MP-2134

to

K.K.).

436

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Y.

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M.

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K.

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M.

Kiyoduka, A

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D.

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Quantum

Bio-Informatics

IV

eds. L.

Accardi,

W.

Freudenberg

and

M.

Ohya

World

Scientific

Publishing

Co.

(pp.

437-450)

NETZCOPE:

A

TOOL

FOR

DISPLAYING

AND

ANALYZING

COMPLEX

NETWORKS

MICHAEL J.

BARBER

Department

of

Foresight and Policy Development,

Austrian

Institute

of

Technology

LUDWIG

STREIT

BiBoS,

Univ. Bielefeld and CCM, Univ.

da

Madeira

OLEG STROGAN

Kyiv,

NaUKMA,

Faculty

of

Informatics

Networks

are

a

natural

and

popular

mechanism

for

the

representation

and

inves-

tigation

of

a

broad

class

of

systems.

But

extracting

information

from

a

network

can

present

significant

challenges.

We

present

NetzCope,

a

software

application

for

the

display

and

analysis

of

networks.

Its

key

features

include

the

visualiza-

tion

of

networks

in

two

or

three

dimensions,

the

organization

of

vertices

to

reveal

structural

similarity,

and

the

detection

and

visualization

of

network

communities

by

modularity

maximization.

1.

Introduction

Networks describe

the

structure

and

dynamics

of

relations

between

objects

or

agents: in

short,

networks

are

everywhere.

In

biology

they

have become

a

prominent

subject

of

research,

in

engineering

network

concepts have be-

come ever

more

important-from

Kirchhoff's laws

in

the

19th

century

to

optimizing

the

design

of

a microchip

to-day-and

in

the

socio-economic

field

"globalization" is

paradigmatic

for

their

dominance. A

short

list

of

examples

may

serve

to

underscore

this

point:

Neural

Networks

The

well-studied

nematode

Caenorhabditis el-

egans

has

a

neural

network

of

some 300

neurons

with

some 7000

connections

between

them.

Metabolic

Networks

metabolic

processes

in

the

cell.

Protein

interaction

networks

physical

interactions

between

an

437

438

organism's proteins.

Transcriptional

networks

regulatory interactions between dif-

ferent genes.

Food

webs

Who

eats

whom?

Sexual

relations

and

infections

AIDS epidemiological models

Pollination

networks

plants

and

their

pollinators

Electric

networks

stability

of power grids

Electronic

networks,

computer

chips

computing speed

Airline

networks

service efficiency

Internet

search engines

Linguistics

words linked by co-occurrence, language families

Social

networks

identification of central players,

gate

keepers,

Collaborations

actors, authors, research labs,

...

Typically these networks exhibit considerable complexity

and

more of-

ten

than

not

their

structure

is far from

transparent.

In

this work, we present NetzCope, a software application for

the

display

and

analysis of complex networks. NetzCope is a general purpose tool for

investigating networks, allowing

the

user

to

interactively explore networks,

especially

with

regard

to

visualizing

the

most

important

relationships in

the

network.

1.1.

Bipartite

Networks

The

NetzCope software was originally developed

to

find

and

display

the

structure

hidden in long lists of

tens

of

thousands

of collaborative research

projects sponsored by

the

European

Union. Said networks are bipartite,

with

links always connecting members of two different sets. Some examples:

Regulatory

networks

transcription

factors

and

target

genes

Economic

networks

Financial centers

and

multinationals, firms

and

board

members

Collaboration

networks

actors

and

films in which

they

appeared

together, laboratories

and

joint

research projects, scientists

and

joint

publications,

...

1.2.

Modularity

Of

particular

interest

to

the

exploration of

the

network of EU-funded

projects-

and,

indeed,

to

networks in

general-is

any possible

modular

structure

of

the

network. Quoting from

an

article on network biology:

Cellular functions

are

likely

to

be

carried

out

in

a highly

modular

manner.

In

general,

modularity

ref

ers

to

a

group

of

physically

or

functionally linked molecules (nodes)

that

work

together

to

achieve

a (relatively)

distinct

function. Modules

are

seen

in

many

systems,

for example, circles

of

friends

in

social networks

or

websites

that

are

devoted

to

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