Table Of ContentPersPeCtIve
Communicative & Integrative Biology 5:6, 519–526; November/December 2012 © 2012 Landes Bioscience
Finding and defining the natural automata acting in living plants
Toward the synthetic biology for robotics and informatics in vivo
Tomonori Kawano,1,2,3,4,* François Bouteau2,3,5,6 and Stefano Mancuso2,3,4
1Faculty and Graduate School of Environmental Engineering; The University of Kitakyushu; Kitakyushu, Japan; 2LINV@Kitakyushu Research Center;
Kitakyushu, Japan; 3LINV@Firenze; Department of Plant Soil and Environmental Science; University of Florence; Sesto Fiorentino, Italy; 4Paris Interdisciplinary
Energy Research Institute (PIERI); Paris, France; 5University Paris Diderot; Sorbonne Paris Cité; Institut des Energies de Demain (IED); Paris, France; 6Institut
de Biologie des Plantes; Bât 630; Orsay, France
The automata theory is the math- automaton), the computational prob-
ematical study of abstract machines lems can be properly solved. A finite-state
commonly studied in the theoretical automaton (FSA), known as the finite-
computer science and highly interdisci- state machine is a behavioral model used
plinary fields that combine the natural to design the computer programs, which is
sciences and the theoretical computer composed of a finite number of states asso-
science. In the present review article, ciated to transitions. A transition is a set
as the chemical and biological basis for of actions allowed starting from one state
natural computing or informatics, some and ending in the same or another state,
plants, plant cells or plant-derived mol- which is started by a triggering event or
ecules involved in signaling are listed and condition. In biology and artificial intel-
classified as natural sequential machines ligence researches, finite-state machine are
(namely, the Mealy machines or Moore sometimes used to describe the neurologi-
machines) or finite state automata. By cal systems.
defining the actions (states and transition In the present review article, as the
functions) of these natural automata, the chemical and biological basis for natural
similarity between the computational computing or informatics, some plants,
data processing and plant decision-mak- plant cells or plant-derived molecules
ing processes became obvious. Finally, behaving as natural sequential machines
Keywords: aqueous computing, automa- their putative roles as the parts for plant- or automata are listed and classified as
ton, plant signaling, plant enzyme based computing or robotic systems are natural sequential machines (namely the
discussed. Mealy machines or Moore machines) or
Abbreviations: AMAs, aromatic mono-
FSA. By defining the actions (states and
amines; DFA, deterministic finite autom-
Introduction transition functions) of these natural
ata; FSA, finite-state automaton; H O ,
2 2 automata, the similarity between the com-
hydrogen peroxide; HO•, hydroxyl radi-
Natural computing is the recently grow- putational data processing and plant deci-
cals; HRP, horseradish peroxidase; IAA,
ing field of research that investigates sion-making processes became obvious.
indole-3-acetic acid; NFA, nondetermin-
models and computational techniques Finally, their putative roles as the parts for
istic finite automata; O•-, superoxide
2 inspired by nature and, dually, attempts to plant-based computing or robotic systems
anion radicals; ROS, reactive oxygen
understand the world around us in terms are discussed.
species; SA, salicylic acid
of information processing.1 It is a highly Handling and manipulating the life.
Submitted: 07/10/12 interdisciplinary field that combines the As natural computing studies attempt to
Revised: 08/09/12 natural sciences and the theoretical com- understand the world including the bio-
puter science. The automata theory is the logical systems arrond us, some approaches
Accepted: 08/10/12
mathematical study of abstract machines may be similar to those taken by synthetic
http://dx.doi.org/10.4161/cib.21805 commonly studied in the theoretical biologists. For understanding the life, it is
computer science.2 Using these abstract simpler and far easier to create a minimal
*Correspondence to: Tomonori Kawano;
Email: [email protected] machines called automata (singular, model that functions as a life or to develop
www.landesbioscience.com Communicative & Integrative Biology 519
much more simplified models mimicking hosts, namely the cells and organisms, Volatile Memory and FSA in Plants
at least a part of life, which is a funda- in which they operate.10 Their key design
mental idea common to newly emerging features, inspired by computer science By considering an intact plant as an indi-
system biology and synthetic biology.3 and engineering, are programmability, vidual sequential machine, the signal
Synthetic biologists engineer complex arti- modularity and versatility. While still processing (perception and transduction)
ficial biological systems to investigate the the works are in progress, the state of the inside the living plant can be attributed to
natural biological phenomena for a variety art “DNA-based biocomputer” now cal- the automaton-like decision-making pro-
of applications.4 Kurihara et al.5 argued culates a square root.11 In the near future, cesses. The most notable case is the action
that the construction of protocells, from due to robust growing of the area, bio- of Venus flytrap (Dionaea muscipula)
a materials-point of view, is important in computers will eventually enable disease which possesses an active trapping mecha-
understanding the origin of life. diagnosis and treatment with single-cell nism to capture small insects.14 Each of
A major goal of synthetic biology is to precision, lead to “designer” cell func- five to seven leaves on this plant has two
develop a deeper understanding of bio- tions for biotechnology, and bring about parts, the upper and the lower leaves,
logical design principles from the bot- a new generation of biological measure- which coordinately capture the insects.
tom up, by building circuits and studying ment tools.10 The trapping action by Venus flytrap has
their behavior in the living cells.6 Since three unique features attracting experts
the ability to quickly and reliably engi- Mealy Machines and Moore not only in biology but also in chemistry15
neer many-component systems from Machines: Two Different Types and physics,16 namely, (1) it has one of the
libraries of standard interchangeable of Sequential Machines in vivo fastest movements in the plant kingdom,
parts is one hallmark of modern tech- (2) it exhibits a “decision-making intelli-
nologies, nowadays, the synthetic biolo- As the machine meets an input, it makes gence” to determine, from a semi-closed
gists insist that we should adapt many a jump to another state, according to the state, whether to proceed to be completely
of established frameworks for describing transition functions defined. By defining closed or fully open, and (3) the Venus
the existing engineered devices to the the actions (states and transition func- flytrap has a “memory” of touch that two
study and manipulation of biological tions) of these natural automata, the simi- consecutive mechanical stimuli within
objects.7 To answer a question if simple larity between the computational data about 30 sec are usually necessary to trig-
biological systems can be built from processing and plant decision-making ger the trap closing. As the sensory organs,
standard, interchangeable parts and processes became obvious. Here, we would three sensitive trigger hairs are protrud-
operated in living cells, synthetic biol- like to compare two types of sequential ing from the upper leaf epidermis of the
ogy is now expanding and affecting the machines, namely, Mealy machines and Venus flytrap, adjacent to the red antho-
educational sceneries. In the last decade, Moore machimes. cyanin pigment that attracts the insects.
the International Genetically Engineered By definition, Mealy machine is repre- According to recent studies, touching of
Machine Competition (iGEM) has been sented formally by sextuple, viz., a three the trigger hairs (by insects) rapidly acti-
offering the chances for students and different sets of Q, Σ, and Δ, two functions vates the mechano-sensitive ion channels
young researchers of various backgrounds δ, and λ, and the initial state q .2,12 Note and generates receptor potentials, which
0
to cope with each other to achieve that (1) Q is a finite set of states; (2) Σ is a induce an action potential required for
designing and assembling of biological finite set of input symbols; (3) Δ is a finite trap closure. Recently, Volkov et al.17 dis-
devices required for building the “genetic set of output symbols; (4) δ is the state covered that closing of the traps can be
machines,” by using a library of standard- transition function which determines the also induced upon electrical stimulation.
ized parts known as BioBricks.8 Standard next state q (ϵ Q) based on the combination The Venus flytrap can accumulate small
biological parts, such as BioBricks, may of the present state p (ϵ Q) and the input a sub-threshold charges, and when the
provide the foundation for a new engi- (ϵ Σ), that is, δ (p, a) = q; (5) λ is the output threshold value is reached, the trap closes.
neering discipline that enables the design function which determines the output b (ϵ Thus, repeated stimulation by smaller
and construction of synthetic biological Δ) based on the combination of the present charges are counted and summed up to
systems with a variety of applications in state p (ϵ Q) and the input a (ϵ Σ), that is, λ induce the closure of the leaves. Therefore,
bioenergy, new materials, therapeutics, (p, a) = b; and (6) q (ϵ Q) is the start state, the researchers have argued that this plant
0
and environmental remediation.9 that is, the state of the machine before any has “volatile memory” for either mechani-
input has been processed. cal touching or electrical stimulation.
Computing with Biological Similarly to a Mealy machine, a Moore By assuming that Venus flytrap itself
Materials machine is also represented formally by is an abstract sequential machine or there
sextuple,13 where Moore machine M = (Q, is an array of sensory cells or molecules
One of the aims in synthetic biology is Σ, Δ, δ, λ, q ). The key difference between equipped within the Venus flytrap, thus,
0
the creation of intelligence based on the the Mealy and the Moore machines are functioning as a sequential machine, the
biological bricks. Biocomputers are man- temporal features of the output as illus- functioning signal “transducer” or “recog-
made biological networks whose goal trated in Figure 1. This point is discussed nizer” processing the mechanical stimuli
is to probe and control the biological in the later section. in Venus flytrap must be interpreted either
520 Communicative & Integrative Biology volume 5 Issue 6
Figure 1. state transitions in Mealy machine and Moore machine. these sequential machines (A) and (B) consist of states (represented by circles),
and transitions (represented by arrows). the initial states are shown by the double arrows. As the machine meets an input, it makes a jump to another
state, according to the transition function defined (based on the current state and the recent symbol of inputs). Above illustrations were adopted from
ref.1 temporal difference in the behaviors of two simple sequential machines are compared in (C) and (D). As shown in (C), Mealy machine’s action is
just to exchange an input event with an output event. In contrast, many signaling molecules may behave similarly to a Moore machine during signal
transduction in aqueous computing or biological systems (D). thus, any given single (chemical) event can be considered as an input for a receptor or
protein involved in signaling. Once the molecule of interest is activated by single (chemical) event such as phosphorylation, binding to calcium, bind-
ing to the ligands, etc., the molecule becomes activated for certain length of time. During the activated state, the molecule (Moore machine) might
keep acting by emitting multiple signals.
as a Mealy machine or Moore machine. encoded by specific genes, thus proteins, in Figure 1, upon receiving an effective
Since a Moore-type sequential machine are preferable. In the below sections, the input signal, a Mealy machine used as a
can be readily converted into a FSA, the nature and the molecular basis for the signaling molecule may emit an output
system conserved in Venus flytrap is now abstract sequential machines or automata signal only once.
described as a FSA M (Fig. 2). naturally equipped in living plants, as the In contrast, a Moore machine may
1
By definition,2 an automaton (M) is interchangeable parts for synthetic biology consecutively emit signals once the tran-
represented formally by a quintuple, M = are discussed. sition to the active state is manifested
(Q, Σ, δ, q , F): where (1) Q is a finite set Signaling Molecules as Moore upon receiving an effective input signal.
0
of states; (2) Σ is a finite set of input sym- machines or Derived FSA. In electriccal Thus, the input signal can be amplified
bols (the alphabet of the automaton); (3) computing models, the Mealy machines by the output through Moore-type sig-
δ is the state transition function (δ: Q × and Moore machines are considered to naling molecules. The example of plant
Σ → Q), which determines the next state be equivalent, thus these machines can be and non-plant signaling molecules act-
q (ϵ Q) based on the combination of the interchangeable.2 However, in the aqueous ing in the manners similar to Moore
present state p (ϵ Q) and the input a (ϵ Σ), computing model or biological models, the machines include photoreceptors such as
that is, δ (p, a) = q; (4) q (ϵ Q) is the start molecular basis for these machines must phytochrome and cryptochrome; many
0
state, that is, the state of the automaton be clearly distinguished. Especially the members of transporters and channel
before any input has been processed; and timing and continuity of the output are proteins such as aquaporins, cation per-
(5) F is a set of final states of Q (i.e., F ⊆ largely different as examples of the behav- meable channels and anion channels;
Q). iors of two simple sequential machines protein kinases such as mitogen-activated
While the Venus flytrap model (Fig. 2) are compared in Figure 1C and D. Here, protein kinase (MAPK), MAPK kinase
deals with an intact plant as an individual this review propose a view that most of (MAPKK), MAPKK kinase (MAPKKK),
FSA, the automata belonging to this type individual signaling molecules found in protein kinase A (PKA), protein kinase
are not available as interchangeable bio- biological systems including plants can C (PKC), plant Ca2+-dependent protein
logical parts to be used out of the living be categorized as the Moore machines or kinase (CDPK), Ca2+/calmodulin-depen-
plants. In order to construct a library of derived FSA. Let’s consider two distinct dent protein kinase (CaMK), and etc.;
interchangeable parts, further search for types of molecules acting as sequential protein-binding and activating proteins
putative molecules behaving as autom- machines placed in the aqueous system or such as calmodulins, trimeric G-proteins,
ata is required. Furthermore, molecules an artificial cell. By definition illustrated samll G protein members and etc.; and
www.landesbioscience.com Communicative & Integrative Biology 521
Figure 2. volatile memory processing determining the closure of the trap in venus flytrap can be attributed to “automata.” (A) the simplified signaling
mechanism of trap closure induced after processing the mechanical input in venus flytrap, supported by experimental and theoretical analyses (Modi-
fied from volkov et al.13). (B) transition state of FsA M counting the number of stimuli. the states allowed in M are represented by circles, and the
1 1
transitions are represented by the arrows. the initial state is shown by the double arrow and the final state is shown with the double circle. Input can
be accepted (thus, closure induced) only after repeated stimuli. FsA M = (Q, Σ, δ, q , F), where Q = {p, q, r}, Σ = {0, 1}, δ(p, 0) = p, δ(p, 1), = q, δ(q, 0)
1 1 1 1 01 1 1 1 1 1 1
= q, δ(q, 1), = r, δ(r, 0) = p, δ(r, 1), = r, q = p, F = r. (C) the behavior of FsA M can be attributed to two types of metaphorical FsA M and M function-
1 1 1 01 1 1 p1 c1
ing as a whole plant and the cells composing the plant, respectively. At the level of molecular interactions, the function for M can be considered as
c1
synthesis of functions for various molecular FsA (M , M ’, M ”…).
m1 m1 m1
DNA-binding proteins acting as tran- the roles of plant peroxidases in the plant glycoproteins, induction of intracellular
scription factors. signaling events involving salicylic acid signaling pathway such as the synthesis of
In case of action plants and locomo- (SA), aromatic monoamines (AMAs) SA and activation of MAPK cascade, or
tive cells of green algae, cytoskeletons and and indole-3-acetic acid (IAA), brought activation of systemic acquired resistance
motor protein complexes controlled under in the below section clearly suggest that associated with systemic propagation of
signaling events can be considered as the this type of molecules can participate the oxidative burst.
Moore machines too. In the above models, the signaling pathways in plants, thus In plants, peroxidases achieve a great
intermediate signals such as phosphorylat- deserve being listed in additions to the deal of oxidation reactions essential for
ing events and releases of secondary mes- Moore-type molecules and FSA from the cells, using H O as an acceptor of
2 2
sengers such as cyclic AMP and calcium plants. e- and a variety of substrates as e- donors,
ions can be considered both as the input The production of reactive oxygen spe- as the group of pioneering researchers of
and output signals for individual Moore cies (ROS), chiefly superoxide anion radi- plant peroxidase in Geneva metaphori-
machines or FSA involved in cellular sig- cals (O•-), hydrogen peroxide (H O ), and cally described that plant peroxidases pos-
2 2 2
nal transduction. hydroxyl radicals (HO•) at the cell sur- sess more functions than a ‘Swiss army
Plant Peroxidase as Redox-Mealy face, well known as the “oxidative burst” knife’.19 Indeed, highly diversified func-
Machines. Despite the above section is one of the earliest events detectable dur- tions of plant peroxidases including regu-
focused on the signal amplifying roles ing the incompatible interactions between lation of H O level, oxidation of various
2 2
for the Moore-type signaling mole- plants and pathogens.18 To date, multiple substrates, generation of ROS (coupled to
cules, it should be also noted that plant roles of ROS have been proposed in direct oxidation of IAA, amines and SA) have
enzymes are rich sources for the Mealy- microbicidal actions, strengthening of cell been reported to date.20 Oxidation of SA
type molecules. The given examples with wall through oxidative cross-linking of is one of the key functions of the ‘knife’.20
522 Communicative & Integrative Biology volume 5 Issue 6
In Figure 3, byproducts of peroxidase-cat-
alyzed oxidation of SA and IAA coupled
to generation of O•- are summarized.
2
Previously, possible overall interactions
between SA and plant peroxidases have
been documented,21,22 and the estimated
reactions for the generation of O•- are as
2
follows:
[1] Native protein (3) + H O →
2 2
Compound I (5) + H O
2
[2] Compound I (5) + SA → Compound
II (4) + SA•
[3] Compound II (4) + SA → Native
protein (3) + SA•
[4] 2 SA• + 2 O → 2 SA+ + 2 O•-
2 2
where SA• and SA+ are free radical
species and the two-electron oxidized
Figure 3. Behavior of plant peroxidase as a redox-active mealy machine (M). Based on the
intermediate product derived from SA, 2
language (input signals) used, M can be separately described as two different automata (M’ and
respectively. Numbers in the small brack- M”). (A) the hourglass model su2mmarizing the superoxide generating reactions catalyzed b2y
2
ets indicate the formal oxidation states plant peroxidases responsive to both salicylic acid (sA, a model substrate for peroxidase cycle),
of the heme. In the reactions above, SA aromatic monoamines (AMA) and indole-3-acetic acid (IAA, a model substrate for oxygenation
behaves as an e- donor while H O acts as cycle).20 (B) redox-active Mealy machine M2’. M2’ = (Q2’, Σ2’, Δ2’, δ2’, λ2’, q02’), where Q2’ = {q0, q3, q4, q5}
2 2 Σ’ = {0, IAA, O}, Δ’ = {0, 1}, δ’(q, 0) = q, δ’(q, IAA) = q, δ’(q, O) = q, δ’(q, 0) = q, δ’(q, IAA) =
the e- acceptor. Then the released SA• may 2 2 2 2 0 0 2 0 4 2 0 2 0 2 4 4 2 4
q, δ’(q, O) = q, δ’(q, 0) = q, δ’(q, IAA) = q, δ’(q , O) = q , δ’(q , 0) = q , δ’(q , IAA) = q , δ’(q,
react with O to form O •-. As O •- can be O4) =2 q4, λ’2(q, 03) = 20, λ3’(q, IA0A) 2= 03, λ’(q, O)5 = 20, λ3 ’(q2, 0) =0 0, 2λ’(5q , IAA) 5= 02, λ5’ (q, O), = 50 , λ2’(q5,
2 2 2 2 5 2 0 2 0 2 0 2 2 4 2 4 2 4 2 2 3
readily converted to H O , one cycle of 0) = 1, λ’(q, IAA) = 0, λ’(q, O) = 0, λ’(q, 0) = 0, λ’(q, IAA) = 0, λ’(q, O) = 0, q ’ = q. (C) redox-
2 2 2 3 2 3 2 2 5 2 5 2 5 2 02 0
SA-oxidizing peroxidase reaction started active Mealy machine M.” M” = (Q”, Σ,” Δ”, δ,” λ”, q ”), where Q” = {q, q, q}, Σ” = {0, HO,
2 2 2 2 2 2 2 02 2 0 1 2 2 2 2
sA}, Δ” = {0, 1}, δ” (q, 0) = q, δ”(q, HO) = q, δ”(q, sA) = q, δ”(q, 0) = q, δ”(q, HO) = q, δ”(q,
with single unit of H O results in yield 2 2 0 0 2 0 2 2 1 2 0 0 2 1 1 2 1 2 2 1 2 1
2 2 sA) = q, δ”(q, 0) = q, δ”(q, HO) = q, δ”(q, sA) = q, λ”(q, 0) = 0, λ”(q, HO) = 0, λ”(q, sA) = 0,
of two units of O •- equivalent to two 2 2 2 2 2 2 2 2 2 2 2 0 2 0 2 0 2 2 2 0
2 λ”(q, 0) = 0, λ”(q, HO) = 0, λ”(q, sA), = 1, λ”(q, 0) = 0, λ”(q, HO) = 0, λ”(q, sA) = 1, q ” = q.
units of H O , thus ROS members are N2ote1, Δ’ = Δ”2 = {01, 1}2 =2 {ϕ, O·-}2. 1 2 2 2 2 2 2 2 2 02 0
2 2 2 2 2
amplified. Experimental evidence in sup-
port of the production of SA• species has
been obtained from an ESR study using and without additional globin radicals, where S and P are the substrate and
ascorbate as a sensitive spin trapper.22 respectively. Interestingly, like plant product of its one-electron oxidation,
Effect of SA on the peroxidase oxidation enzymes (Compound I), the ferryl inter- respectively.20
state was carefully examined through mediates of human hemoglobin oxidize IAA can be oxidized by plant peroxi-
spectroscopic analysis using horseradish aromatic monoamines (AMAs), leading dases (chiefly by HRP in model experi-
peroxidase (HRP) as a model enzyme to the production of AMA radicals and ments) by this mechanism but there is no
and the hourglass model presented in O •-,29 through the catalytic cycle so- strict substrate specificity in this conven-
2
Figure 3 was proposed.23,24 In place of called pseudo-peroxidase cycle. tional H O-dependent peroxidase cycle.20
2 2
SA in the model, AMAs can be alter- Metabolism of IAA, the principal The plant peroxidases including HRP oxi-
native mediator of peroxidase reaction form of auxin in higher plants, is of great dize IAA also via the H O-independent
2 2
releasing O •-,24-26 with some exception.23 interest to plant biologists. Plant peroxi- pathway requiring molecular oxygen (O ),
2 2
The redox cycles of plant peroxidases are dases are considered to be involved in the since most peroxidases of plant origins
largely analogous to those found in other metabolism of IAA, by oxidizing IAA via (but not animal or microbial origins)
hemoproteins. In the hourglass model two different mechanisms: a conventional are considered to be highly specific IAA
(Fig. 3A), the overall inter-conversions H O-dependent pathway and one that oxygenases, which possess the domains
2 2
among the native form, ferrous form, requires O but not H O .30-32 The con- structurally similar to a specific motif in
2 2 2
Compounds I, II and III, and irrevers- ventional peroxidase cycle for the oxida- auxin-binding proteins.30 The proposed
ibly inactivated form (P-670) of plant tion of various substrates coupled to the reaction cycle for IAA oxidation is initi-
peroxidase are summarized. Compounds consumption of H O proceeds as follows: ated via the formation of a ternary com-
2 2
I and II of plant enzymes are considered [1] Native protein (3) + H O → plex,31 enzyme-IAA-dioxygen, yielding
2 2
to possess the hemes at ferryl states with Compound I (5) + H O IAA cation radicals and O•- as by-prod-
2 2
and without additional porphyrin radi- [5] Compound I (5) + S → Compound ucts as follows:32
cals, respectively.27,28 Thus, Compounds I II (4) + P [7] E + IAA ↔ [E-IAA],
and II are analogous to the ferryl hemo- [6] Compound II (4) + S + H+ → Native [8] [E-IAA] + O ↔ [E-IAA-O ],
2 2
globin intermediates from human with protein (3) + H O + P [9] [E-IAA-O ] → E + IAA•+ + O•-,
2 2 2
www.landesbioscience.com Communicative & Integrative Biology 523
where E and IAA•+ stands for enzyme of plant peroxidase inhibitors such as biology researchers, Paramecium species
and IAA cation radicals, respectively. salicylhydroxamic acid or binding of car- are very familiar laboratory tools for cell
Thus, plant peroxidases are capable of bon monooxide to heme pockets of the biological and environmental studies.39
catalyzing the IAA-dependent generation enzymes results in the loss of catalytic Cells of Paramecium species are now
of O•- in the absence of H O . However, activity. Therefore, the output from the considered as model systems for study-
2 2 2
the nature of the enzyme during forma- inactivated state can be considered as 1. ing cellular signal transduction mecha-
tion of enzyme-substrate complexes such nisms, obviously equipped with minimal
as [E-IAA] and [E-IAA-O ] has not been In Vivo Cellular Computing decisin-making propeties.40 Since signal
2
identified.20,32 Furthermore, this model and Plantoids perception, processing and reactions are
does not explain an additional action of completed within these unicellular organ-
IAA as a suicide substrate against plant Above sections have indicated the simi- isms, some researchers have described the
peroxidases by which the enzymes are larity between the computational digital cells of Paramecium species as “swimming
irreversibly inactivated.33 Previously, it data processing and plant decision-mak- sensory cells”41 or “swimming neurons.”42
has been suggested that the oxidation ing processes by defining the actions It is well known that Paramecium spe-
statuses of HRP intermediates34 and soy- (states and transition functions) of the cies including the photosynthetic species,
bean peroxidase intermediates35 formed natural automata in plant systems. Today, green paramecia (Paramecium bursaria),
in the presence of IAA, namely, [E-IAA] our approaches for synthetic biology migrate toward the anodic electrode when
and [E-IAA-O ], may be ferrous enzyme targeting the bio-inspired computing exposed to an electric field in a medium.43
2
and the O -bound form of the enzyme and robotics have yet started. The idea This type of cellular movement is known
2
(Compound III) in the oxygenase cycle of of constructing plantoids (named after as the galvanotaxis. In fact, over a century
peroxidases, respectively. Therefore, IAA analogy to androids and humanoids) was ago, it has been known that Paramecium
can be used as a tester substrate for show- proposed by the group of Prof. Mancuso species exhibit galvanotaxis in which the
ing the presence of oxygenase cycle-depen- (Univ. Florence, Italy). The plantoids are ciliate cells align with an electric field or
dent O•--generating pathway (Fig. 3A). by definition, the plant-inspired robots voltage gradient and swim toward the
2
Interestingly, the transitions among proposed for the investigation of both anode if the electric field is sufficiently
different oxidation states in plant peroxi- biological and technological issues.36,37 strong.44 Recently, a pharmacological
dases can be interpreted as the actions of Development of technologies applicable study has revealed that green paramecia
Mealy machines. The models involving for such novel area of robotics is highly is responsive to the electric field and the
IAA (Fig. 3B) and SA (Fig. 3C) were challenging topics to be achieved. It is electric stimulus applied to green para-
shown as different redox Mealy machines. also challenging to develop the intelli- mecia is converted to a galvanotactic cel-
Not like the Moore machines, the mem- gent plant-inspired or plant-related robots lular movement with the involvement
bers of the Mealy machines require highly equipped with plant-derived automata, of the T-type calcium channels on the
repeated inputs for performing the sub- by connecting computer and natural plasma membrane.43 The above studies
stantially available levels of outputs by or artificial plant cellular networks, for indicate the possibility for finely geared
consecutively repeating the input-output enhanced data processing. neuronal controls and engineering of uni-
cycles. Actually that is the case for plant At present, we merely assume that the cellular micro-machineries. In fact, the
peroxidases catalyzing a number of cycles operational performance of plantoids galvanotactic responsiveness observed in
of O•--generating reactions to achieve the can be designed based on the designs of Paramecium species (particularly P. cau-
2
signaling upon repeated consumption of abstract sequential machines or automata, datum) has attracted the attention of
substrates such as SA and IAA. thus, the plantoids may possibly function bioengineers in the fields of biorobotics,
after installing the interchangeable parts microrobotics or BioMEMS (biological
Dark Logic Models Using derived from plant molecules or cells. micro-electro-mechanical systems) in
Inhibitors Converts Therefore, in Figure 4A-C, automata at order to develop electrically controllable
the Biological Mealy Machines different levels were proposed for future micro-machineries.45-47 Furukawa and
into Moore Machine development of plantoids, namely, plan- his colleagues have suggested that in vivo
toid automata, cellular automata and cellular robotics using the cells of green
Discussion in the above section sug- molecular automata. paramecia as micro-machines control-
gested that many of plant enzymes such Possible natural automata for robotic lable under electrical and optical stimuli,
as peroxidases can be considered as natu- application found in other biological has a variety of engineering applications
ral Mealy machines. However, the use of systems. In the present article, we have such as transport of micro-sized particles
speficic inhibitors targetting the enzymes focused on the finding and defining the in the capillary systems.48,49 In fact Moore
may behave as the inputs for the Moore natural automata acting in living plants. machines and derived FSA determining
machines when dark logic was employed. Our ongoing study also suggests that nat- the direction of cellular migration (both
For an instance, by considering the pres- ural automata such as Moore-type autom- galvanotactic and phototactic move-
ence of active enzyme as 0 and loss of ata or FSA can be found and defined in ments) in the cells of green paramecia can
activity as 1 (thus, dark logic), addition living cells of protozoa. To many of cell be defined (data not shown).
524 Communicative & Integrative Biology volume 5 Issue 6
Perspectives: Deterministic finite
automata (DFA) and nondeterministic
finite automata (NFA). Lastly, the authors
wish to discuss the difference between the
deterministic finite automata (DFA) and
nondeterministic finite automata (NFA)
to be applied in designing the plantoids or
study in the in vivo cellular robotics.
Many of readers with biological back-
grounds may wonder if the biological
systems function as machines for infor-
matics. Can every events and outcomes
observed in the biological systems be
considered clearly as the series of digi-
talized information expressed with 1 or
0, as discussed in the above sections? In
fact, the answer is yes with some cautions.
It is apparently obvious that the applica-
tions of automata in the biological topics
require special cautions in order to repro-
duce the real biological phenomena. In
nature, especially in biology, accuracy of
the automata may largely vary from cell
to cell, from molecule to molecule; thus,
an experimentally forced input “1” to
Figure 4. Proposed models for plantoids, cells, and molecules functioning as automata. (A) A
the automata in biological system could
model of plantoid produced by Prof. s. Mancuso (Univ. Florence). (B) Image of an electro-physi-
be resulted in the corresponding outputs ologically monitored cell (Prof. F. Bouteau, Univ. Paris-Diderot). (C) An artificial enzyme behaving
with statistically determined range of as a plant peroxidase mimic.38 (D) state transitions in deterministic finite automata (DFA) M. M =
3 3
variance. This is largely due to the fact (Q, Σ, δ, q , F), where Q = {q, q, q, q}, Σ = {0, 1}, δ(q, 0) = q, δ(q, 1) = q, δ(q, 0) = q, δ(q, 1)
3 3 3 03 3 3 0 1 2 3 3 3 0 0 3 0 1 3 1 0 3 1
= q, δ(q, 0) = q, δ(q, 1) = q, δ(q, 0) = q, δ(q, 1) = q, q = q, F = {q, q}. (E) state transitions in
that there are both diversity and redun- 2 3 2 3 3 2 2 3 3 3 3 3 1 03 0 3 2 3
nondeterministic finite automata (NFA) M’. M’ = (Q’, Σ’, δ’, q ’, F’), where Q’ = {r, r, r}, Σ’ = {0,
dancy in the sets of sensory or signaling 3 3 3 3 3 03 3 3 0 1 2 3
1}, δ’(r, 0) = {r}, δ’(r, 1) = {r, r}, δ’(r, 0) = ϕ, δ’(r, 1) = r, δ’(r, 0) = r, δ’(q, 1) = ϕ, q ’ = r, F’ = r.
molecules and/or cells differed in thresh- 3 0 0 3 0 0 1 3 1 3 1 2 3 2 2 3 2 03 0 3 2
olds to the signal intensities or quanti-
9. Anderson JC, Dueber JE, Leguia M, Wu GC, Goler
ties. One of the likely answers to describe References JA, Arkin AP, et al. BglBricks: A flexible standard
and/or design the plant-derived autom- 1. Kari L. The many facets of natural computing. for biological part assembly. J Biol Eng 2010; 4:1;
Commun ACM 2008; 51:72-83; http://dx.doi. PMID:20205762; http://dx.doi.org/10.1186/1754-
ata is the modification of the automata org/10.1145/1400181.1400200. 1611-4-1.
models from DFA to NFA, although this 2. Tomita Y, Yokomori T. Automata and formal lan- 10. Benenson Y. Biocomputers: from test tubes to live
idea is yet to be tested through future guage. 1992; Morikita Publ. Tokyo. cells. Mol Biosyst 2009; 5:675-85; PMID:19562106;
3. Amos M. Genesis machines. New science of biocom- http://dx.doi.org/10.1039/b902484k.
researches. Figure 4 (D and E) com- puting. Atlantic Books 2006; London. 11. Benenson Y. Biocomputing: DNA computes
pares the DFA to NFA with equivalent 4. Andrianantoandro E, Basu S, Karig DK, Weiss R. a square root. Nat Nanotechnol 2011; 6:465-7;
processing capability. The latter model Synthetic biology: new engineering rules for an PMID:21814231; http://dx.doi.org/10.1038/
emerging discipline. Mol Syst Biol 2006; 2:2006, nnano.2011.128.
(Fig. 4E) allows two different responses 0028; PMID:16738572; http://dx.doi.org/10.1038/ 12. Mealy GH. Method for synthesizing sequential cir-
upon input in the initial state. This type msb4100073. cuits. Bell Syst Tech J 1955; 34:1045-79.
5. Kurihara K, Tamura M, Shohda K, Toyota T, Suzuki 13. Moore EF. Tgedanken-experiments on sequential
of lowered stringency could be achieved
K, Sugawara T. Self-reproduction of supramolecu- machines. In: Automata studies. Princeton Univ.
by involvement of multiple cells or mol- lar giant vesicles combined with the amplification Press, Princeton, USA. 1956; pp. 129-153.
ecules in the signaling events, thus the of encapsulated DNA. Nat Chem 2011; 3:775- 14. Volkov AG, Adesina T, Markin VS, Jovanov E.
81; PMID:21941249; http://dx.doi.org/10.1038/ Kinetics and mechanism of Dionaea muscipula
resultant performance of the automata nchem.1127. trap closing. Plant Physiol 2008; 146:694-702;
would be much closer to the naturally 6. Nandagopal N, Elowitz MB. Synthetic biology: PMID:18065564; http://dx.doi.org/10.1104/
observable phenomena. integrated gene circuits. Science 2011; 333:1244- pp.107.108241.
8; PMID:21885772; http://dx.doi.org/10.1126/sci- 15. Ueda M, Nakamura Y, Okada M. Endogenous fac-
ence.1207084. tors involved in the regulation of movement and”
Acknowledgements 7. Canton B, Labno A, Endy D. Refinement and stan- memory” in plants. Pure Appl Chem 2007; 79:519-
dardization of synthetic biological parts and devices. 27; http://dx.doi.org/10.1351/pac200779040519.
This work was supported by a Grants-in-
Nat Biotechnol 2008; 26:787-93; PMID:18612302; 16. Li Y, Zhang M. Nonlinear dynamics in the trapping
Aid for Scientific Research by Ministry of http://dx.doi.org/10.1038/nbt1413. movement of the Venus flytrap. Proc. Am Control
Education, Culture, Sports, Science and 8. Brown J. The iGEM competition: building with Conf. 2011; pp. 3514-351.
biology. Synth Biol 2007; 1:3-6; http://dx.doi. 17. Volkov AG, Adesina T, Jovanov E. Closing of venus
Technology (MEXT), Japan (Research org/10.1049/iet-stb:20079020. flytrap by electrical stimulation of motor cells. Plant
Project Number: 23656495). Signal Behav 2007; 2:139-45; PMID:19516982;
http://dx.doi.org/10.4161/psb.2.3.4217.
www.landesbioscience.com Communicative & Integrative Biology 525
18. Yoshioka H, Bouteau F, Kawano T. Discovery of oxi- 27. Rutter R, Valentine M, Hendrich MP, Hager LP, 37. Mancuso S. The roots of plant intelligence. 2010;
dative burst in the field of plant immunity: Looking Debrunner PG. Chemical nature of the porphyrin pi http://www.ted.com/talks/stefano_mancuso_the_
back at the early pioneering works and towards the cation radical in horseradish peroxidase compound roots_of_plant_intelligence.html.
future development. Plant Signal Behav 2008; 3:153- I. Biochemistry 1983; 22:4769-74; PMID:6313048; 38. Kagenishi T, Yokawa K, Kadono T, Uezu K, Kawano
5; PMID:19513209; http://dx.doi.org/10.4161/ http://dx.doi.org/10.1021/bi00289a024. T. Copper-binding peptides from human prion pro-
psb.3.3.5537. 28. Schulz CE, Rutter R, Sage JT, Debrunner PG, tein and newly designed peroxidative biocatalysts. Z
19. Passardi F, Cosio C, Penel C, Dunand C. Peroxidases Hager LP. Mössbauer and electron paramagnetic Naturforsch C 2011; 66:182-90; PMID:21630593;
have more functions than a Swiss army knife. Plant resonance studies of horseradish peroxidase and its http://dx.doi.org/10.5560/ZNC.2011.66c0182.
Cell Rep 2005; 24:255-65; PMID:15856234; http:// catalytic intermediates. Biochemistry 1984; 23:4743- 39. Miyoshi N, Kawano T, Tanaka M, Kadono T, Kosaka
dx.doi.org/10.1007/s00299-005-0972-6. 54; PMID:6093863; http://dx.doi.org/10.1021/ T, Kunimoto M, et al. Use of Paramecium species in
20. Kawano T. Roles of the reactive oxygen species- bi00315a033. bioassays for environmental risk management: deter-
generating peroxidase reactions in plant defense and 29. Kawano T, Pinontoan R, Hosoya H, Muto S. mination of IC values for water pollutants. J Health
50
growth induction. Plant Cell Rep 2003; 21:829-37; Monoamine-dependent production of reactive oxy- Sci 2003; 46:429-35; http://dx.doi.org/10.1248/
PMID:12789499. gen species catalyzed by pseudoperoxidase activity jhs.49.429.
21. Kawano T, Sahashi N, Takahashi K, Uozumi N, of human hemoglobin. Biosci Biotechnol Biochem 40. Pech LL. Regulation of ciliary motility in Paramecium
Muto S. Salicylic acid induces extracellular super- 2002; 66:1224-32; PMID:12162542; http://dx.doi. by cAMP and cGMP. Comp Biochem Physiol A
oxide generation followed by an increase in cyto- org/10.1271/bbb.66.1224. 1995; 111:31-7; http://dx.doi.org/10.1016/0300-
solic calcium ion in tobacco suspension culture. The 30. Gazaryan IG, Lagrimini LM, Ashby GA, Thorneley 9629(95)98516-J.
earliest events in salicylic acid signal transduction. RN. Mechanism of indole-3-acetic acid oxida- 41. Machemer H, de Peyer JE. Swimming sensory cells:
Plant Cell Physiol 1998; 39:721-30; http://dx.doi. tion by plant peroxidases: anaerobic stopped-flow electrical membrane parameters, receptor proper-
org/10.1093/oxfordjournals.pcp.a029426. spectrophotometric studies on horseradish and ties and motor control in ciliated protozoa. Verh.
22. Kawano T, Muto S. Mechanism of peroxidase actions tobacco peroxidases. Biochem J 1996; 313:841-7; Deutsche Zool Gesellschaft, Erlangen, 1977; pp.
for salicylic acid-induced generation of active oxygen PMID:8611164. 86-110.
species and an increase in cytosolic calcium in tobac- 31. Savitsky PA, Gazaryan IG, Tishkov VI, Lagrimini 42. Naitoh Y. Protozoa. In: Electrical Conduction and
co cell suspension culture. J Exp Bot 2000; 51:685- LM, Ruzgas T, Gorton L. Oxidation of indole- Behaviour in “Simple” Invertebrates, Ed. Shelton
93; PMID:10938860; http://dx.doi.org/10.1093/ 3-acetic acid by dioxygen catalysed by plant peroxi- G.A.B., Clarendon Press, Oxford, 1982; pp. 1-48.
jexbot/51.345.685. dases: specificity for the enzyme structure. Biochem J 43. Aonuma M, Kadono T, Kawano T. Inhibition of
23. Kawano T, Muto S, Adachi M, Hosoya H, Lapeyrie F. 1999; 340:579-83; PMID:10359640; http://dx.doi. anodic galvanotaxis of green paramecia by T-type
Spectroscopic evidence in support of horseradish per- org/10.1042/0264-6021:3400579. calcium channel inhibitors. Z Naturforsch C 2007;
oxidase compound II-catalyzed oxidation of salicylic 32. Kawano T, Kawano N, Hosoya H, Lapeyrie F. Fungal 62:93-102; PMID:17425113.
acid but not of phenylethylamine. Biosci Biotechnol auxin antagonist hypaphorine competitively inhib- 44. Ludloff K. Untersuchungen uber den
Biochem 2002; 66:651-4; PMID:12005064; http:// its indole-3-acetic acid-dependent superoxide gen- Galvanotropismus. Arch Gesamte Physiol 1895;
dx.doi.org/10.1271/bbb.66.651. eration by horseradish peroxidase. Biochem Biophys 59:525-54; http://dx.doi.org/10.1007/BF01789963.
24. Kawano T, Muto S, Adachi M, Hosoya H, Lapeyrie Res Commun 2001; 288:546-51; PMID:11676477; 45. Itoh A. Motion control of protozoa for Bio MEMS.
F. Spectroscopic evidence that salicylic acid con- http://dx.doi.org/10.1006/bbrc.2001.5800. IEEE/ASME Trans Mechatron 2000; 5:181-8;
verts a temporally inactivated form of horseradish 33. Kawano T, Kawano N, Lapeyrie F. A fungal auxin http://dx.doi.org/10.1109/3516.847091.
peroxidase (compound III) to the irreversibly inac- antagonist, hypaphorine prevents the indole-3-acetic
46. Ogawa N, Oku H, Hashimoto K, Ishikawa M. Motile
tivated verdohemoprotein (P-670). Biosci Biotechnol acid-dependent irreversible inactivation of horserad-
cell galvanotaxis control using high-speed tracking
Biochem 2002; 66:646-50; PMID:12005063; ish peroxidase: inhibition of Compound III-mediated
system. Proc IEEE Int Conf Robotics Automation
http://dx.doi.org/10.1271/bbb.66.646. formation of P-670. Biochem Biophys Res Commun
2004; pp. 1646-51.
25. Kawano T, Pinontoan R, Uozumi N, Miyake C, 2002; 294:553-9; PMID:12056802; http://dx.doi.
47. Ogawa N, Oku H, Hashimoto K, Ishikawa M.
Asada K, Kolattukudy PE, et al. Aromatic mono- org/10.1016/S0006-291X(02)00513-2.
Dynamics model of paramecium galvanotaxis for
amine-induced immediate oxidative burst lead- 34. Kawano T, Furuichi T, Muto S. Controlled free
microrobotic application. Proc IEEE Int Conf
ing to an increase in cytosolic Ca2+ concentration salicylic acid levels and corresponding signaling
Robotics Automation 2005; pp. 1258-63.
in tobacco suspension culture. Plant Cell Physiol mechanisms in plants. Plant Biotechnol 2004;
48. Furukawa S, Karaki C, Kawano T. Micro-particle
2000; 41:1251-8; PMID:11092910; http://dx.doi. 21:319-35; http://dx.doi.org/10.5511/plantbiotech-
transporting system using galvanotactically stimu-
org/10.1093/pcp/pcd052. nology.21.319.
lated apo-symbiotic cells of Paramecium bursaria. Z
26. Kawano T, Pinontoan R, Uozumi N, Morimitsu Y, 35. Takayama A, Kadono T, Kawano T. Heme redox
Naturforsch C 2009; 64:421-33; PMID:19678550.
Miyake C, Asada K, et al. Phenylethylamine-induced cycling in soybean peroxidase: Hypothetical model
49. Furukawa S, Kawano T. Enhanced microsphere
generation of reactive oxygen species and ascorbate and supportive data. Sens Mater 2012; 24:87-97.
transport in capillary by conditioned cells of green
free radicals in tobacco suspension culture: mecha- 36. Mazzolai B, Laschi C, Dario P, Mugnai S, Mancuso
paramecia used as living micromachines controlled
nism for oxidative burst mediating Ca2+ influx. Plant S. The plant as a biomechatronic system. Plant Signal
under electric stimuli. Sens Mater 2012; 24:375-86.
Cell Physiol 2000; 41:1259-66; PMID:11092911; Behav 2010; 5:90-3; PMID:20023403; http://
http://dx.doi.org/10.1093/pcp/pcd053. dx.doi.org/10.4161/psb.5.2.10457.
526 Communicative & Integrative Biology volume 5 Issue 6
