Plant neurobiology by uup

Prof K N GaneshaiahCoordinator, IBIN

Head, Department of Forestry and Environment Sciences,

University of Agricultural Sciences, Bangalore -

Mention of advances in Human brain research

Written by:

Human Brain

What about plants?

Is there anything called ‘Neurobiology’ for Plants?

and YOU will answer at the end

Listen to me for next 45 minutes

Science is ever changing truth

DNA/Protien as genetic material

Human feelings

arise from heart

Earth centric theory

Atom and

God’s particles

Journey of Plant Neurobiology till date…

Study of Plant behaviour

Plant physiology and Biochemistry

Scientists for the birth of Plant Neurobiology

Charles Darwin (1809-1882) Sir Jagadish C. Bose (1858-1937)

By

Udachappa U. PujarI Ph.D

UHS13PGD41

Plant Neurobiology: in search of plant neurons and brain

I SeminarOn

Definitions Neurobiology: Neurobiology is the study of cells of the

nervous system and the organization of these cells into functional circuits that process information and mediate behaviorNervous system may be defined as an organized

constellation of cells (neurons) specialized for the repeated conduction of an excited state from receptor sites or from other neurons to effectors or to other neurons

Plant Neurobiology: Branch of plant science deals with signaling and behavior of plants and parts/systems responsible for it

Events

Signal passed

Signal perception by sensory organs

Signal transmission by neurons and

neurotransmitters

Signal processing and decision making

Signal to body organs to for reaction

How human neurobiology works?

Now questions arise……..!

• How this signal perception takes place in plants?

• Do plants exhibit neurons, neurotransmitters and brain as signal processing centers?

Do plants smell?

Do plants hear sound?

Do plants communicate each other?

Can plants do mathematics

Do plants keep memory

Do plants can see?

Plant behaviour

Equipments used in studies

CrescographMicroscopeCamerasIsotopesSensorsPhytotronsComputers and softwares

Case studies on Plant Behavior

Do plants smell?

Plants smell recognize volatile compounds produced by neighboring plant as signal for switching on their specific metabolic activity

Figure 1: Volatile Organic Compounds (VOCs) emitted by injured plants have a specific ratio and concentration of components. (A). The danger signals emitted by the family provide warning that a species-specific enemy (specialist) is nearby. In contrast, plants that receive a VOC message from other families might elicit a general defense response to prevent damage by herbivores (generalists) attacking various plant species (B). By sharing common VOC information across the plant kingdom, plants are able to prevent attack from a broad range of herbivores.

(Hirokazu et al., 2012)

(A) foraging toward a 20‑day‑old tomato plant,

Figure 2: Seedling of Cuscuta pentagona(Mark et al., 2006 )

(B) attaching to and beginning to grow from stems of tomato seedlings and

(C) close up of C.. pentagona attachment.

α‑pinene, β‑myrcene, and β‑phellandrene

So plant do smell

Inference:

Do plants respond to sound?

Figure 3: Behavioral response of young roots of Zea mays to a continuous 220 Hz sound coming from left field (white arrow).

Mention of Solomon Island’s tribal activity

Video showing Solomon Islands

So plant respond to sound

Inference:

Can plants keep memory?

Figure 4: Somatic homologous recombination in UV-C- and flg22-treated plants. a, Schematic representation of a recombination substrate used for monitoring somatic homologous recombination (lines IC1 and IC9). GUS, b-glucuronidase gene; Hpt, hygromycin-resistance gene. Homologous region is shown in dark blue. b, Recombination events (blue spots highlighted by black arrows) giving a measure of homologous recombination frequency (HRF; Homologus recombination frequency) in line IC1 after flg22 treatment. Scale bar, 1 mm; inset, £3 original magnification. c, Somatic HRF in untreated and UV-C-treated S0 plants. Results are means ^ s.e.m. (n . 50 plants; t-test *P , 0.05). d, Somatic HRF in either untreated plants, plants treated with flg22 A. tum., or treated with flg22. Results are means ^ s.e.m. (n . 40 plants; t-test *P , 0.05). (Jean et al., 2006)

Figure 5: Somatic HRF in S0 plants and in the next four generations. S0 plants (line IC1) were either untreated or UV-treated. Somatic HRF was measured in untreated S1, S2, S3 and S4 plants. Results are means ^ s.e.m. (n . 50 plants; t-test *P , 0.05 compared with the corresponding S0 -UV generation).

(Jean et al., 2006)

Figure 6 : Somatic HRF in either self-pollinated or out crossed plants. a) Somatic HRF in offspring of either self-pollinated untreated plants, UV-treated plants, or

plants in which one of the parents was UV-treated (n . 40 plants; t-test *P , 0.05). b) Somatic HRF in offspring of either self-pollinated untreated plants (white bar), flg22-treated

plants (grey bars), or plants in which one of the parents was flg22-treated (hatched bars; n . 35 plants; t-test *P , 0.05).

All the results are means ^ s.e.m.

(Jean et al., 2006)

Figure 7: Somatic HRF in either self-pollinated or out crossed plants. Somatic HRF in offspring of plants in which one parent was wild type (WT) and the other harbored the recombination substrate (n . 40 plants; t-test *P , 0.05). White bar, both parents untreated; dark hatching, both parents UV-treated; light hatching, one parent UV-treated. All the results are means ^ s.e.m.

(Jean et al., 2006)

So plants do keep memory

Inference:

Can plants do mathematics?

Figure 8: Starch content levels from experiments with unexpected variation in either starch content at the onset of darkness or the time of onset of darkness. (A) Starch turnover in Arabidopsis grown in 12-hr light/12-hr dark, then subject to unexpected early (8 hr, n = 6 individual rosettes, circles) normal (12 hr, n = 6, squares) or unexpected late (16 hr, n = 5, triangles) onset of darkness. (B) Starch turnover in Arabidopsis cca1/lhy mutant grown in 12-hr light/12-hr dark, then subject to unexpected early (9 hr, circles), or normal (12 hr, squares) onset of darkness (n = 6–10).

(Antonio et al., 2013)

Figure 9: Starch content levels from experiments with unexpected variation in either starch content at the onset of darkness or the time of onset of darkness.(C) Starch turnover in Arabidopsis exposed to different daytime light levels: 90 μmol quanta m−2 s−1 (open squares) or 50 μmol quanta m−2 s−1 (filled squares) (both n = 5, previously all plants grown in 12-hr light/12-hr dark with 90 μmol quanta m−2 s−1). (D) Starch turnover in Brachypodium grown in 12-hr light/12-hr dark, then subject to unexpected early (8 hr, circles) or normal (12 hr, squares) onset of darkness (both n = 6). Error bars are standard error of the mean throughout.


Figure 10: Chemical kinetic models capable of implementing analog arithmetic operations. (A) Pictorial summaries of schemes for analog implementation of addition, subtraction and multiplication between the concentrations of two molecules S and T. Square brackets indicate concentrations. (B) and (C) Schematic behavior of the stromal concentrations of S and T molecules ([SC] and [TC] respectively), in (B) first and (C) second arithmetic division models. In the first model, the T molecule tracks the time to expected dawn after a reset-time tr. In the second model the T molecule concentration increases with time proportionally to 1/(expected time to dawn) between tr1 and tr2. (D) and (E) Pictorial summaries of (D) first and (E) second analog arithmetic division models (not all reactions shown in pictures, for full details see ‘Materials and methods’). In the reaction schemes, molecules not attached to the starch granule surface have a ‘C’ subscript. The blue disk represents components of the starch degradation apparatus potentially activated by the S molecule in the first model, and by the ST complex in the second model. (Antonio et al., 2013)

Figure 11. Chemical kinetic models capable of implementing analog arithmetic operations. A. Model 1 B. Model 2 arithmetic division models to Arabidopsis


So plants can do mathematics

Inference:

Do plants communicate?

Plant Communication

Within plant Between plants

With insect/microb

es

Through chemicals Through Electric signals

Figure 12: Mature leaves detect changes in CO2 concentration and elicit a stomatal response in developing leaves. a, Leaf-cuvette experiment. Plants of Arabidopsis (Columbia, Col-0) were grown for 4 weeks under ambient CO2 (360 p.p.m.) until leaf insertions 5 to 13 had developed. These mature leaves were enclosed in transparent airtight cuvettes under CO2 concentrations of either 720 or 360 p.p.m. Subsequent leaf insertions developed outside the cuvette under ambient CO2. Plants were maintained in cuvettes for 7 to 9 days until the next five leaf insertions had matured, the last three of which were investigated for stomatal density (no. of stomata per mm2) and index ((no. of stomata/no. of stomata & no. of epidermal cells)2100).

Figure 13: Left → Stomatal index and density for new leaves (insertions 16 to 19) under ambient CO2 when mature leaves (insertions 5 to 13) inside cuvettes are supplied with increased CO2 (720 p.p.m.). Both stomatal density and index are reduced in new leaves if the supply of CO2 is increased to the mature leaves. Right → Reverse experiment: mature leaves inside cuvettes are under CO2 at 360 p.p.m.; external CO2 is 720 p.p.m. Stomatal density and index increase in response to the decreased CO2 around the mature leaves. ***P*0.0005; *P*0.05; bars, s.e.m.; n4150. (Lake et al., 2001)

Figure 14: Effect on stomatal index of new leaves of reducing light incident on mature leaves by using neutral density filters (shade) or transparent filters (full light). Stomatal index of new leaves is reduced when mature leaves are shaded. ***P*0.0005; *P*0.05; bars, s.e.m.; n4150.

(Lake et al., 2001)

Figure 15: Schematic representation of the custom-designed experimental unit (not in scale). (a) The seal at the base of the central cylindrical box ensured that chilli seeds arranged in a circle around the adult plant were chemically isolated from it. (b) All seeds and adult plants within a replicate unit were housed within 2 different sized square boxes, one inside the other, with the air in between the two boxes removed using a vacuum pump. The whole experimental unit was custom-made in colourless cast acrylic material (ModenGlas), which transmitted 92% of visible light, but was opaque to ultraviolet and infrared wavelengths. (Monica et al., 2012)

Figure 16: Early growth of chilli seedlings depends on the presence and identity of their neighbour. (a) Seedlings growing next to a fennel (grey solid line and triangles) are marginally significantly taller than those growing next to an adult chilli plant (black solid line and squares; Pair-wise contrasts, P = 0.07) and significantly taller than seedlings in the empty control (black dotted line and white diamonds; Pair-wise contrasts, P = 0.01). The observed differences in above-ground growth among treatments (adult fennel plant, grey solid line and triangles; adult chilli plant, black solid line and squares; empty control, black dotted line and white diamonds) are amplified over time. Only plants that emerged by day 14 are included in these analyses (n = 32 per treatment). Error bars indicate standard errors. (b) Growth differences disappear when seedlings are allowed to grow in the absence of any adult plant after emergence (n = 80 per treatment). Error bars indicate standard errors. (Monica et al., 2012)

Figure 17: Mean final root size of chilli seedlings is affected by the presence and identity of their adult neighbours. (a) Overall, maximum root length differed significantly depending on the neighbouring plant present in the sealed central box (n = 32 per treatment). Seedlings growing next to adult chilli plants had significantly shorter roots than those in the empty control or growing with the fennel (P = 0.015). (b) The presence of a neighbouring fennel during germination and emergence caused an increase in early root development of chilli seedlings when the communication channels are blocked, but not when unblocked (light grey bars) (F masked . F open and Control masked; P = 0.027; n = 80 per treatment). Differences disappeared when seedlings were allowed to grow away from a fennel plant (dark grey bars) (P = 0.94; n = 80 per treatment). Error bars indicate standard errors. (Monica et al., 2012)

Let us watch something now….

Wait!!!This isn’t just an animated movie, it has something

scientific

This was applied in a Hollywood block buster

So plants can do communicate

Inference:

Much understanding of these behaviors

Through study of plant neurobiological view

Deals with animal/human like neurons, synapses, neurotransmitters and the brain

Similarities between animal/human neurobiology with plant neurobiology

Some of the representatives of these subsystems are proposed for three levels of organisation of increasing complexity, cell, organ, and organism in plants (P and Roman script) and animals (A and italic script) (Barlow, 1999; Miller and Miller, 1995). Arrows (←and →) indicate that the process is delegated, respectively, to either a lower or higher organisational level.

Table 1: According to J.G. Miller’s Living System Theory (Miller, 1978) there are 10 subsystems out of a total of 20 subsystems which process information



Plant Neurons

Figure 18: Vascular bundles throughout the plant body. Thin strands of vascular tissue form networks in leaves, join into bundles in shoots, and transform into a large central cylinder of roots which is encircled by pericycle and endodermis

Still a hypothetical view : Vascular bundles as plant neuronsXylem: NeuronPhloem: Axon Reasons:

The word neuron derived from greek language, which means vegetable fiber

Only channel throughout the plant body which passes any information

Transmits ion to different part of plant organs

Phloem perform functions similar to axon as passing signals form xylem to the tissue or cells

Neurons are cells that are specialized to receive, propagate, and transmit electrochemical impulses.

(Baluska et al., 2009)

Plant SynapsesCellular end poles as plant synapses

Plant synapses are stable actin-supported adhesive domains, assembled at cellular end-poles (cross-walls) between adjacent plant cells of the same cell file, across which auxin and other chemical signals are transported from cell to cell via F-actin-driven and brefeldin A sensitive vesicular trafficking pathways

(Baluška et al. 2003 ; Barlow et al. 2004).

Figure 19: Possible scenario for evolution of epithelial and auxin transporting plant synapses. Under repeated pathogen attacks and with progressive exposures of ancient terrestrial plants to dry environments, ancient surface epithel-liketissue (A) developed in to the contemporary epidermis and endodermis plant epithels (B). Finally,auxin-transporting synapses evolved from epithelial synapses (C). Red lines show synaptic cell cell adhesion domains; red crosses depict APB1; yellow balls are recycling vesicles; and orange dots represent auxin. (Baluska and Stafeno, 2013)

Figure 20: Developmental auxin-transporting plant synapse and its role in gravisensing. (a) In axial plant organs such as roots, cross-walls (synapses) transport auxin from cell-to- cell; the recycling of the putative auxin efflux carrier PIN1 (blue) and the auxin influx carrier AUX1 (red) is essential for this process. (b) Several molecules have been localized to these auxin-transporting plant synapses. (c) Because of gravity force, the protoplast exerts a greater mechanical load on the physical bottom of any cell in axial plant organs (indicated by the larger size of the blue arrow). This asymmetric protoplastic load is effectively balanced by robust cell walls maintaining tubular cell shapes.

Figure 21: Developmental auxin-transporting plant synapse and its role in gravisensing. (c) Because of gravity force, the protoplast exerts a greater mechanical load on the physical bottom of any cell in axial plant organs (indicated by the larger size of the blue arrow). This asymmetric protoplastic load is effectively balanced by robust cell walls maintaining tubular cell shapes. (d) The absence of this protective force would result in a distortion of the protoplast shape because of the preferential accumulation of protoplastic masses at the physical bottom. (e) A differential mechanical load exerted on the plasma membrane domains, which constitute the plant developmental synapse, results in a high plasma membrane tension experienced at the physical bottom, which inevitably facilitates more exocytic events and less endocytic events at the blue (PIN1-enriched) plasma membrane domain, whereas the opposite situation is encountered at the red (AUX1-enriched) domain. This inherently asymmetric nature of plant synapses, encompassing both molecular and physical aspects, results in the polar transport of auxin along the gravity vector. (Frantisek et al., 2005)

Plant NeurotransmittersA chemical substance which is released at the end of a nerve fibre by the

arrival of a nerve impulse and, by diffusing across the synapse or junction, effects the transfer of the impulse to another nerve fibre, a muscle fibre, or some other structure.

Table 2: Discovery of neurotransmitters

Sources: (Kruk and Pycock 1990; Roshchina 1991, 2001; Kuklin and Conger 1995; Oleskin 2007; Kulma and Szopa 2007)

Table 3: Level of neurotransmitters in living organisms

Sources: Fernstrom and Wurtman 1971; Kruk and Pycock 1990; Hsu et al.:1986; Roshchina 1991, 2001; Oleskin et al. 1998; Tsavkelova et al. 2000)

Figure 22: The scheme of the evolution in the neurotransmitter (biomediator) function(Mariela and Christina, 1997)

Table 4: The established functions of neurotransmitters in living organisms

Table 5: Processes and functions in plants modulated by acetylcholine and biogenic monoamines

Even plant cells produce electric signals in an emergency

Action Potentials (AP)

Variable Potentials (VP)

Local electrical Potentials (LEP)

Plant Electric Signals

Animal/Human brain or neurons or cells produce electric signals in an emergency

Table 6: Comparison of basic characteristics of APs, VPs, and SPs` (Matthias et al., 2009)

Figure 23:. Techniques for measuring electrical signals in plants. (a) Extracellular recording with four channels and a reference electrode inserted in the soil. , electrical stimulation. An AP (right) generated by electrical stimulation appeared successively at electrodes 1, 2, 3 and 4. (b) Intracellular measurement of the membrane potential with a microelectrode inserted into the cytoplasm of an algal cell while the reference electrode is in contact with the artificial pond water (APW) outside the cell. Both electrodes are filled with KCl, clamped in Ag/AgCl pellet holders and connected to an electrometer. (Jorg and Slike, 2007)

Figure 24: Techniques for measuring electrical signals in plants. (c) Phloem potential measurements; an aphid in feeding position with its stylet inserted into a sieve element on the upper side of a leaf. (d) After the aphid is separated from its stylet by a laser pulse, the stylet stump exuded sieve tube sap to which the tip of a microelectrode was attached. Cooling the shoot evoked an AP transmitted acropetally within the phloem, while flaming of a leaf generated a VP with different form and of long duration. t, time.

(Jorg and Slike, 2007)

Figure 25: Electrical signalling in Mimosa pudica. (a)When the tip of a leaf pinna is stimulated by spontaneous cooling with ice water or mechanically by touch, an AP is evoked and transmitted basipetally within the rhachis with a speed of 20–30 mm s-1. The tertiary pulvini at the base of the leaflets respond to the AP, causing ion and water fluxes that lead to leaf movements. This type of signal stops at the base of the pinna, and no further transmission occurrs. (b)When the leaf is stimulated by cutting, a basipetally moving VP is generated in the rhachis, irregular in shape and long in duration. Its speed is slower (5–6 mm s-1) than that of the AP; however, it is able to pass through secondary pulvini at the base of the pinna and causes leaflet movements of neighbouring pinna, and also bending of the primary pulvinus at the base of the petiolus.

(Jorg and Slike, 2007)

Figure 26: Photosynthetic response of electrical signaling in poplar. (a) Experimental arrangement of gas exchange recordings. The plant was heat stimulated for 3 s by the flame of a lighter at the base of a mature leaf (*) to evoke a VP . (b) Typical response of JCO2 and gH2O of the opposite, right leaf at a distance of 15 cm upon flaming of the left leaf. The arrow denotes the instant of injury. At 180 s after stimulation, the net CO2 uptake rate (black graph) decreased immediately and then recovered almost completely after 900 s, while the gH2O (grey graph) remained stable. (Jorg and Slike, 2007)

Figure 27: Photosynthetic response of electrical signaling in poplar. (c) Spatio-temporal changes of DF/F′m assessed by chlorophyll fluorescence imaging. The image area (length, 22 mm; width, 17 mm) covers the center of a leaf while the opposite leaf was stimulated by flaming (*) at a distance of 15 cm similar to the set-up shown in (a). Times are given in relation to the instant of injury (at time 0). Changes in DF/F′m took 240 s to become apparent.A false colour shift from blue to yellow in the intervein area, equivalent to a reduction of DF/F′m from 0.6 to about 0.2, indicates the decrease of photosynthesis. The bar translates the false colour code into values of DF/F′m.

Jorg and Slike, 2007

Table 5: Well-documented physiological effects of electrical signals in plants

Jorg and Slike, 2007

Plant Brain

In plants root transition zone act as brain

Each root apex is proposed to harbor brain-like units of the nervous system of plants. The number of root apices in the plant body is high, and all ‘‘brain units’’ are interconnected via vascular strands (plant neurons) with their polarly-transported auxin (plant neurotransmitter), to form a serial (parallel) neuronal system of plants. (Alpi et al., 2007)

Figure 28: Anatomical basis of root and shoot apices. Anatomical organization of root (a) and shoot (b) apices. Note very regular cell files, with cross-walls representing plant synapses, in root apices. On the other hand, cells in shoot apices are irregularly shaped and fail to arrange into regular cell files (Frantisek et al., 2005)

Figure 29: Sensory zones in the root apex. There are two clearly defined sensory zones in the root apex: the root cap covering the meristem and the transition zone interpolated between the meristem and elongation region. Both these sensory zones receive diverse signals and the output is differential switch-like onset of rapid cell elongation, resulting in either straight growth (when all post mitotic cells start their rapid cell elongation simultaneously) or rapid turnings of the root apex. The transition zone is flooded with sucrose, which allows energy demanding ‘brain-like’ information processing in cells of the transition zone

(Frantisek et al., 2005)

Similarities of human brain with root tip transition zone

Human/Animal brain– ATP consumption: 104

molecules of ATP to transmit 1bit of information

at a chemical synapse – Consumes 20% of body

oxygen– Very well safeguarded by

skull– Presence of otoliths to in

ear to sense gravity

Plant brain (Root tip transition zone

– In plants ATP consumption: Highest at root

– Oxygen consumption: Highest at root tansition

– Very well safeguarded by root cap

– Presence of statoliths in root transition zone

Applications

Biorobotics: Plantoids

Terrafarming

Nutreint sensors in soils

Diversity studies

Figure 1: A schematic view of the loop for the formulation of a formal model of a biological system, by experimental validation.

(Barbara et al., 2010)

Now

Is there anything called as ‘Plant Neurobiology’?

Gregor Johann Mendel(1822-84)

Father of Modern Genetics!!!

• 1856: Series of breeding experiments to study

plant heredity

• First to work out the basic laws governing the

inheritance to genes

• 1866: Published detailed account of his findings

before the Brunn Natural History

• His work was not accepted by Scientific

community

• 1884: Death of Mendel

• 1900: Mendel’s work re-emerged by the

independent works of Hugo Devries, Tschermark

and Correns

Take home message

Plant neurobiology – Still an infant

The hypotheses has to proved – Molecular genomics and

cell biology, chemical and biochemical ecology

Needs a combined effort of plant scientists from diverse

backgrounds and from all disciplines

Results in better understanding of crop plants

What more you want??

By Udachappa U. Pujar

I Ph.DUHS13PGD41

Thank you