1

Life on the edge or

Salt lake

As sesile organisms plants must live wherever they happen to germinate

And they usually manage

2 LT50 -10.40C LT50 -5.70CLT50 -9.40C LT50 -4.80C

3

Irritability-SensitivityIrritability-Sensitivity

Irritability is one of the major defining characteristics of all living organisms – the ability to respond to the environment

Urggh!!Yum!!!

respond to Change --> Evolve

4

Sensing and Responding to Environment

• A minimum of 300 genes are required to produce a bacterial cell (based on the number of genes in Mycoplasma).

Escherichia coli contain 3000 genes. The apparently inessential 2700 (90%!) are thought necessary to enable the bacteria to tolerate randomly fluctuating environmental variation

• Many of these genes are involved with the specification of signaling components.

• Signaling, in its broadest sense, is now the major preoccupation of plant and environmental sciences research, including Biotech companies.

5

Mendel is currently licensing WeatherGard™ genes. WeatherGard™ genes are regulatory genes that allow plants to be engineered to be more tolerant to drought, freezing, and high salt soils. Thus, they are "proof of concept" that regulatory genes control valuable agricultural traits.

Crops with WeatherGard™ genes can grow normally under low moisture conditions and resist frost damage. This not only increases crop yields and grain quality, but also expands the available area suitable for sustainable agriculture. WeatherGard™ crops may also reduce the need for irrigation water. The potential value of drought and frost tolerance is huge. As little as a 1% increase in grain production due to better drought and frost tolerance will generate $3 to $4 billion per year.

Mendel Biotech is a pioneer in the application of functional genomics in plants. Mendel's mission is to discover and characterize the function of plant regulatory genes and to develop high-value products

6

• the Weathergard genes which are regulatory TFs, of the CBF/DREB type (C-repeat-binding factor/dehydration responsive element-binding factor 1), concentrated on the "promoter" to switch on tolerance traits at the proper times. If the gene was constantly over-expressing the drought or cold-resistant trait, then the plant would not grow normally, making it commercially useless.

So far, no plants that don't have the Weathergard gene, have been found .

7

Question?

• How do cells communicate? inside (intracellular) and outside (extracellular) connentions?

• By phones.

“cellular”

• A molecule in a biological system passes information in its shape,

charge, hydrophobicity, and reactivity. Any change in its

composition encodes new information in the amount, rate, and

duration and where in the cell or organism the change takes place

8

Factors that determine plant stress responses

different signals are thought to have their own receptors which initiate a downstream signaling cascade

9

The Signalling ProblemsThe Signalling Problems

Cells are exposed to a lot of signals from environment (light, temperature, ions, O2, chemicals or water stress)Signals can be from different chemical classes:small organic molecules, lipids, peptides, gases: how can the cell know which signals are the real ones? Sorting out the relevant signals from the irrelevantSorting out the relevant signals from the irrelevant

Detecting signals at low concentrationsDetecting signals at low concentrations

Translating diverse signals into a Translating diverse signals into a common ‘language’common ‘language’

10

Some General Points

• Single signal may start several cascades

• Signal may be tiny, response can be big

• Many signals are received at the same time – and

may activate / repress the same cascade

• “Crosstalk” can occur (one cascade influences

another)

• Not all cells in an organ are sensitive to same

signals (e.g. stomata more sensitive to ABA than

other cells in the leaf). Receptors must be present to

activate the response pathway.

11

Properties of effective ReceptorsProperties of effective Receptors

to be classified as a receptor (rather than just a binding protein) several criteria must be fulfilled

Specificity – a receptor must be able to distinguish between often closely-related signals

High affinity – signals are often present in low concentrations – effective receptors can often detect nM to pM concentrationsSaturability – a cell has a finite number of receptors and, thus there is a limit to the number of ligand molecules a cell can bind

Reversibility – ligand-receptor association is not covalent – as the ligand concentration drops the complex can dissociate

Coupling – the receptor transfers a signal from ligand to cell

this last feature, more than any other distinguishes a receptor from a binding protein

12

Some SolutionsSome Solutions

Sorting out the [ir]-relevant signals

Detecting signals at low concentrations

Receptors with a high degree of specificity

Receptors with high affinity coupled to an amplification

Activation of signalling pathways designed around a limited number of common processes

13

Signalling the Easy WaySignalling the Easy Way

If you have a signal that is freely permeable across the plasma membrane and that is produced in (relatively) large amounts then signalling is remarkably simple….

Nucleus

Cortisol or brassinosteroids (steroids) can cross the membrane where it binds to the receptor that is also a transcription factor and on binding the hormone it migrates to the nucleus and activates transcription of specific genes.

Steroidhormone

GR

However the majority of signalling systems aren’t that simple

TF

TF

14

Signal transduction in plants

http://fig.cox.miami.edu/~cmallery/150/memb

15

• Hormones can coordinately induce many genes in a pathway

• ROS can induce many genes via their effects on the TFs

(GOTO ROS damage & signaling.ppt)

• Organelle, Mitochondria has been proposed to act as integrator of stress responses, especially important for assesing the severity of stress

Coordination of stress responses

16

Potential Sensors for Abiotic Stress Signals

• Given the multiplicity of stress signals, many different sensors

are expected, although none have been confirmed for the

major environmental fluctuations: cold, drought, or salinity(PC14:s165 Xiong, Schumaker and Zhu)

• However, all above stresses induce transient Ca2+

influx into cell cytoplasm • Ca2+ signals: a Central Paradigm in Stimulus–Response

coupling

• Therefore, channels responsible for this Ca2+ influx may represent one type of sensor for these stress signals.

• Activation of certain Ca2+ channels by cold, or stretching by salt may result from physical alterations in cellular structures.

17

Plasma Membrane• At least two major classes of Ca2+ channels

reside in the plasma membrane. Those that are relatively nonselective with respect to cation and possess a high single-channel conductance are known as maxi-cation channels. The second class is more selective for cation, exhibits a smaller single-channel conductance, and is known as voltage-dependent cation channel 2

• A common feature of most such channels is their activation by membrane depolarization. It is thought that such voltage gating might comprise the critical factor responsible for channel activation during signaling

18

Why Calcium?

• Metabolism in all cells requires orthophosphate (Pi) and phosphorylated organic compounds, particularly for cytosolic reactions associated with transduction of free energy.

• The low solubility product of Ca2+ with Pi would have required the early evolution of mechanisms for maintenance of [Ca2+]cyt at a level well below the

millimolar concentrations that prevail in seawater.

• Thus, transport systems that export Ca2+ from the cytosol are present in all cells to sustain steady state values of [Ca2+]cyt in the submicromolar range.

• This homeostatic mechanism is ideal for subsequent evolution of Ca2+-based signaling pathways.

19

Ca2+ Ca2+

20

Decoding of the Ca2 + signal by conformational changes in EF hand

proteins (Calmodulin=CaM)

Calmodulin is a conserved Ca2+ receptor with a flexible conformation

21

Activation of CDPK through intramolecular binding of the calmodulin-like domain

The junction has autoinhibitor (in red) and a binding site for the calmodulin-like domain (CaM-LD). The CaM-LD is attached by a short tether. The N- and C-term of the CaM-LD each contain a pair of Ca2+-binding EF-hands (the open circles represent those hands with no bound Ca2+). The black bars in the two lobes signify a 'closed' confirmation. An 'open' confirmation results when Ca2+ ions (purple) are bound (signified by movement of the black bar to an exposed surface location). In the apo and basal states, the autoinhibitor interacts with the kinase domain and blocks enzyme activity. Activation occurs when Ca2+ fill all four Ca2+-binding EF-hands in the CaM-LD, causing a conformational change that disengages the autoinhibitor–kinase domain interaction (dotted line). The C-term of the CaM-LD is shown pre-bound to the junction at basal Ca2+ concentrations. The N-term acts as the trigger that binds Ca2+ during a Ca2+ release.

22

Intracelular Ca2+ wave

23

Location of Ca2+ transporters in Arabidopsis

Calcium enters plant cells through Ca2+channels in plasma membranes

In the plasma membrane there are hyperpolarization-activated Ca2+ channels (HACC), depolarization-activated Ca2+ channels (DACC), voltage-insensitive cation channels (VICC), voltage-gated Ca2+ channels, H+/Ca2+-antiporters

1. Plasma membrane,

2. tonoplast,

3. mitochondria, plastids, and other endomembranes.

Low resting levels of cytosolic free Ca2+ are sustained by Ca2+-ATPases and by the Ca2+/H+ antiporter in vacuolar membrane, which remove Ca2+ from the cytosol.

24

Phospholipids as secondary messangers

PIP2: PHOSPHATIDYLINOSITOL 4,5-BIPHOSPHATE

Hydrolysis of PIP2 is activated by different PLC, stimulated either by G-protein or protein tyrosine kinase.

DAG: Diacylglycerol activates the protein kinase C family, that play a crucial role in cell growth and differentiation.

25

HOW Ca2+ SIGNAL is PROPAGATED

Conformational changes in IP3R transduce subsequent

signaling. The distribution of positive and negative charges stabilizes the open conformation of the channel and allows the entry of Ca2+ into the cytoplasm.

External stimulus can trigger Ca2+ influx from INTERNAL stores

The Goal of signaling

pathways is to

produce appropriate

reactions to the type

& strength of

stimulus

Principles of information transmission

processingprocessing

Environmental inputEnvironmental input

reactionreaction

growthgrowth

proliferationproliferation

death death

necrosis necrosis

apoptosis apoptosis

movement movement EFFECTOREFFECTORFUNCTIONSFUNCTIONS

Generate new Generate new informationinformationdistribution of information distribution of information by 2by 2ndnd messengers messengers

Getting the Message AcrossGetting the Message Across

Many signals cannot cross the plasma membrane are detected by transmembrane receptors that transduce the stimulus to the next step in the signalling pathway

Extracellular ligand-binding

domain

Intracellular domain – couples to next step (may have enzymatic

activity)

Transmembrane domain

Plasma membrane

outside

inside

•Seven-transmembrane (serpentine)•Ligands include proteins, peptides,lipids, other small molecules

Signal via heterotrimeric G-proteinsand second messengers

Ligand binding initiates signal via G-protein activation

•Multi-transmembrane•>20 known

Signal via conduction of ions acrossmembranes

Ligand binding initiates channel opening

III. Ion channel receptors

II. RTKs

I. 7tmRECEPTOR SUBTYPES

interact withmembrane receptors

28

The G PROTEIN cycle

Processes found in both plants and animals are indicated in green, processes reported only for plants are in purple and those reported only for animals are indicated in red. GEF (guanine nucleotide exchange factor) and GDI (guanine nucleotide dissociation INHIBITOR) are two G binding proteins so far only identified in animals. In addition to desensitization and internalization, arrestins also act as adapter proteins in the regulation of intracellular SIGNALING. (inset) The round-leaf phenotype exhibited by G (gpa1)

http://stke.sciencemag.org/cgi/content/full/sci;310/5745/71

29

Typical structure of a G protein linked receptor

30

“It is the most targeted family of receptors” by drug manufacturers, it is estimated that a quarter of all pharmaceuticals focus on GPCRs.

31

GTP-binding proteins come in two flavours, small monomeric GTP-binding proteins (e.g. p21ras) and heterotrimeric G proteins. The basic GTP/GDP

binding cycle is the same in both cases.

On-Off Switches – GTP-Binding Proteins

GDP

GTP

Pi

GTPGDP

GDP

ACTIVE

INACTIVE

Exchange of bound GDP for

GTP

Active subunit can interact with and activate the next step in the

signalling pathway

subunit dissociates

from

subunit GTPase

activity GTP>GDP+Pi

subunit reassociates

with

32

Ras (p21ras) is a good example of this type of switch. Ras is a small (21 kDa) monomeric protein that binds GTP or GDP and has intrinsic GTPase activity

p21ras GDP p21rasGTP

p21ras

p21ras GDP

On

Off

GTP

Pi

GDP

ACTIVEINACTIVE

Guanine nucleotide exchange factor interacts with ras

This causes exchange of bound GDP for GTP

Intrinsic GTPase activity hydrolyses GTP to GDP and Pi

Ras GTPase stimulated by

association with GTPase-activating

protein (GAP)

Activated ras interacts with and activates the next component in the

signalling pathway

On-Off Switches – GTP-Binding Proteins

33

Most Small GTPases Are Highly Conserved Regulators of Intracellular

Trafficking

34

Crosstalk between GPCRs and small GTPases A) The binding of agonist to a GPCR promotes GTP exchange on the G subunit, which leads to its dissociation from G subunits. The activated proteins G and G can positively or negatively regulate various downstream effectors. In addition to this ‘classical’ method of signalling, GPCRs can also signal via heterotrimeric G-proteins to Ras (via G) and Rho GTPases (via G12/G13). Gbg proteins stimulate Src-dependent activation of MMPs that release heparin-binding EGF (HB-EGF), which can activate receptor tyrosine kinases (RTK), resulting in Ras activation. G12/G13-mediated RhoA activation involves RhoGEFs, e.g. PDZ-RhoGEF, LARG and p115-RhoGEF

B) Before fMLP receptor activation, the Ral GEF RalGDS is localized to the cytosol and maintained in an inactive complex with b-arrestins. fMLP receptor activation results in the membrane translocation of the b-arrestin–RalGDS. b-Arrestin (Arr) receptor binding is proposed to dissociate the b-arrestin–RalGDS complex freeing RalGDS to activate membrane-bound Ral. Ral activation results in actin cytoskeleton reorganization. (C) Rab5 regulates the endocytosis of AT1AR into clathrin-coated vesicles and mediates the fusion with early endosomes. Rab7 regulates the targeting of AT1AR to lysosomes for degradation, whereas Rab4 and Rab11 regulate the rapid or slow recycling of receptors from early endosomes.

35

Activation of NADPH Oxidase by a small GTPase Rac1

dominant dominant

36

(A) Represents the overview of signaling pathway under stress condition. Stress signal is first perceived by the membrane receptor, which activates PLC and hydrolyses PIP2 to generate IP3 as well as DAG. Following stress, cytoplasmic calcium levels are up-regulated via movements of Ca2+ ions from apoplast or from its release from intracellular sources mediated by IP3. This change in cytoplasmic Ca2+ level is sensed by calcium sensors which interact with their down stream signaling components which may be kinases and/or phosphatases. These proteins affect the expression of major stress responsive genes leading to physiological responses. (B) Early and delayed gene expression in response to abiotic stress signaling. Various genes are triggered in response to stress and can be grouped under early and late responsive genes. Early genes are induced within minutes of stress perception and often express transiently. In contrast, various stress genes are activated slowly, within hours of stress expression and often exhibit a sustained expression level. Early genes encode for the transcription factors that activate the major stress responsive genes (delayed genes). The expression of major stress genes like RD/KIN/COR/RAB18/RAB29B result in the production of various osmolytes, antioxidants, molecular chaperones and LEA-like proteins, which function in stress tolerance.

Generic signal transduction pathway as well as the expression of early and late genes in response to abiotic stress signaling.

37

On-Off Switches – Protein PhosphorylationOn-Off Switches – Protein Phosphorylation

Protein Kinases – transfer phosphate from ATP to specific amino acids

C

C C O

O

H

NH Serine

C

C C O

O

NH

OP

O-

O-

O-Phosphoserine

KinaseATP

ADP

Protein Phosphatases – remove phosphate from specific amino acids

C

C C O

O

H

NH Serine

C

C C O

O

NH

OP

O-

O-

Phosphatase

Pi

Phosphorylation Dephosphorylation

38

Cascading kinases

Protein Kinases often work in a cascade with each being able to activate several molecules.

Result - one signal can activate many molecules

39

Signal amplification

40

G-protein linked receptors interact with kinases

• Very widespread and diverse in functions.• Vision, smell, blood vessel development.• Many diseases work by affecting g-protein linked receptors.• Whooping cough, botulism, cholera, some cancers

• Up to 60% of all medicines exert their effects through G-protein linked receptors.

Given the mammalian paradigm it is surprizing that the Arabidopsis genome encodes only one prototypical G subunit and two (possibly more) G subunits. It is remarkable that given this paucity of G proteins, null mutations of GPA1 and AGB1 are not lethal. These mutations give rise to altered phenotypes but not to reduced plant viability or fertility

Last month (Science 2007) A G Protein-Coupled Receptor Is a Plasma Membrane Receptor for the Plant Hormone Abscisic Acid

Showed that the G protein-coupled receptor genetically and physically interacts with the G protein {alpha} subunit GPA1 to mediates all known ABA responses in Arabidopsis

41

CONNECTING THE RECEPTOR TO RAS

SH2

EGFR

SH3SH3

SH3

ProProProProPro

SosRasRas GDP

Ras is membrane-anchored by a farnesyl moiety

Ras is now accessible

to Sos

RasRas GTP

GDP

42

Signaling from Ras GTPasesSignaling from Ras GTPases

• Extracellular regulated kinase (ERK) cascades are conserved from yeast to humans

• Classical example of transmission from membrane to nucleus

• Cascade of transducers terminates at an effector

Enter nucleus to phosphorylate transcription factor (the TARGET)

MAP kinases(ERKs, JNK, p38)

MAPKKK*

MAPKK

MAPKKK*

MAPKK

MAPK

MAPK

•Activated by Thr/Tyrphosphorylation•Ser-directed•Can translocate tonucleus

•Receive Ras signal at membrane•Ser-directed

•Activated by Serphosphorylation•Thr/Tyr-directed

43

History: cloning the first MAPK Gene (MPK4)

(A) Top: WT, mpk4, and mpk4 expressing activation loop mutated MPK4 (AEF-HA).

The lower panels: adaxial leaf cells

(C) Northern of RNA from wt and mpk4 probed with MPK4 cDNA

(D) The upper panel shows kinase activities immunoprecipitated from mpk4 expressing wt HA-tagged MPK4 and mutated MPK4 (T201A/Y203F; AEF-HA). L er control. The lower panel shows a Western blot of the same immunoprecipitates

The mpk4 mutant has been identified as a dwarf among Ds transposon-tagged lines. Interestingly, mpk4 exhibits constitutive systemic acquired resistance with elevated salicylate levels, increased resistance to virulent pathogens and constitutive PR gene expression, which are common characteristics of many Arabidopsis mutants with elevated resistance. These results suggest that wt MPK4 represses SAR.

44

mkk2 Null and MKK2-Overexpressing Plants Exhibit Opposite Cold and Salt Tolerance

Phenotypes

Freezing treatment was carried out either with previous acclimation (accl.) for 24 hr at +4°C or by directly subjecting the plants to freezing conditions (shock). Pictures were taken 3 days after freezing treatment.

The salt-sensitive phenotype of mkk2 null lines. Germination of wild-type and mkk2 null plants (mkk2) was tested on agar plates with 100 mM or 150 mM NaCl.

45

An Overview of Plant Development

• Signals (environmental cues, photoreceptors, and hormones) affect three fundamental processes:

Cell division

Cell expansion

Cell differentiation

http://aggie-horticulture.tamu.edu/faculty/davies/students/ngo

The Plant life cycle

dormancy germination growth and development flowering and fruiting death

46

Plant responses to environment

• Being rooted in one place plants must respond to changes in environmental conditions / stresses.

• Signals from the surrounding environment are transmitted via Signal Transduction Pathways.

• In guard cells, signal transduction network integrates water status, hormone responses, light, CO2 and other environmental conditions to regulate stomatal aperture.

47

The mechanism of stomatal closure

ABA

Ca2+

ROS

Stomatal closure)K+ efflux, turgor decrease( ?

ABA ↓

5 min

Stomatal pore

The Biochemical approach

48

The Biochemical approach

The effect of H2O2 and Ca2+

on stomatal opening.

Measurements of Ca2+ currents in guard cells

49

ABA activates Ca2+ channels via NADPH oxidaseIn absence of NADPH, ABA does not induce Ca2+ influx ,

)thus ROS production is generated prior to Ca2+ increase( .

The Biochemical approach

50

Genetic approach

ABA does not activate Ca2+ channels in abi mutants.

H202 activates Ca2+ channels in abi1-1 but not in abi2-1

Every activity is executed by a protein (or RNA) that is encoded by a gene

51ROS signal is received by abi1-1 but not in abi2-1

Genetic approach

52

Ca2+ channels are activated by H2O2 and mediate ABA signaling in guard cells leading to stomatal closure

Pei et al., Nature 406, 731-734

rboh

DPI

Oxidase

Stomatalclosure

(PPaseI) (PPase2C)

Guard cells

53

Schroeder et al. 2001 Guard cell signal transduction. In Annu Rev Plant Physiol Plant Mol Biol

אור כחול גורם לפוספורילציה של

משאבת הפרוטונים ושל תעלות

האשלגן

Signaling steps to stomatal opening

54

ABA triggers cytosolic Ca2+cyt increase that signals

to open Vacuolar and Plasma membrane channels

וסגירתן ABAמעורבות בפתיחת שמעורבים ממברנליים חלבונים פעולת ועידוד בעיכובהפיוניות

As vacuoles take up >90% of the guard cell’s volume, the ions exported from the cell must first be transported from vacuoles to cytosol

55

ABA …blocks GA Signal Transduction

GerminationNature (2006) 439, News & ViewsElusive ABA receptor found

GAR

signal transducti

on

ABA ..

…inhibits GA signal transduction,

Recently, the RNA-binding protein FCA, a homologue of an ABA-binding protein ABAP1 (Razem), was identified as an ABA receptor in the regulation of flowering time7. However, ABA receptors involved in seed development, seedling growth and stomatal movement remain elusive.

Nature (2006), October

Fawzi A. Razem

56

ABA receptor isolated

Nature 443, 823-826 (October 2006)

The RNA-binding protein FCA is an abscisic acid receptor Nature 439, 290-294 (January 2006)

Fawzi A. Razem1, Ashraf El-Kereamy1, Suzanne R. Abrams2 and Robert D. Hill1

The phytohormone abscisic acid (ABA) regulates various physiological processes in plants. The molecular mechanisms by which this is achieved are not fully understood. Genetic approaches have characterized several downstream components of ABA signalling, but a receptor for ABA has remained elusive. Although studies indicate that several ABA response genes encode RNA-binding or RNA-processing proteins, none has been found to be functional in binding ABA. Here we show that FCA, an RNA-binding protein involved in flowering, binds ABA with high affinity in an interaction that is stereospecific and follows receptor kinetics. The interaction between FCA and ABA has molecular effects on downstream events in the autonomous floral pathway and, consequently, on the ability of the plant to undergo transition to flowering. We further show that ABA binding exerts a direct control on the FCA-mediated processing of precursor messenger RNA. Our results indicate that FCA is an ABA receptor involved in RNA metabolism and in controlling flowering time.

57

ABA receptor is a G-protein

Guards on hormones. (left) The aperture of a stomatal pore in a leaf is controlled by osmoregulation of two guard cells. When the G protein-coupled receptor GCR2 is inactive, the protein phosphatase ABI1 inhibits stomata closure by attenuating abscisic acid signal transduction. (right) Hyposmotic conditions in guard cells (relative to surrounding tissue) cause stomata to close. This involves activation of GCR2 by ABA, which triggers signaling pathways that lead to closed stomata.

ABA Receptor Found (again)

58

ABA Receptor Found (again)

ION CHANNELS??! It takes a membrane to make sense out of disorder in biology. You have to be able to catch energy and hold it, storing precisely the needed amount and releasing it in measured shares (Lewis Thomas 1974)

Ion channels are membrane protein complexes and they play an essential role in the diffusion of ions across cell membrane.

Why do we need ion channels? Membranes are phospholipid bilayers and they

build a hydrophobic, low dielectric barrier to hydrophilic and charged molecules. Charged molecules or atoms could not penetrate this barrier. Ion channels provide a high conducting, hydrophilic pathway across the hydrophobic interior of the membrane. Specificity

Ion channels are highly specific filters, allowing only desired ions pass through

60

IonomicsProtein pores in the membrane that open or close in response to chemical signals.

Allow or block the flow of ions such as Na+ K+ or Ca2.+

Activated by a ligand on the extracellular side.

Causes a change in ion concentration inside the cell.

Ion-channel Receptors

Membrane potential = 0 < 0

The H+ crowd outside the membrane results in more positively charged outside than inside. This difference is called Membrane potential; it can be used to bring positively charged ions into cells following the gradient

61

Membrane Depolarization & HYPERPOLARIZATION

Normal state, with membrane potential of -65 mV. The yellow band represents a semi-permeable membrane. Red indicates surplus of positive charge, blue indicates surplus of negative charge. Every positive ion has a negative counter-ion, but some of the ion pairs are separated as shown.

Change the extracellular medium to higher concentration of KCl. Potassium ions carry positive charge to the inside, neutralizing some of the excess negative charge.

Throughout this event, an equal amount of positive and negative charge was present in the system. The membrane simply separates an amount of charge that is determined by existing conditions.

Change the extracellular medium to a solution with 0.3 times the normal concentration of potassium chloride. Potassium ions shift out, carrying positive charge to the exterior. Now in the new steady state, there is more displaced charge across the membrane HYPERPOLARIZATION

What is an action potential and how is it generated?

inside

outsideNa+

Na+

K+K+

Na+

Why the need to control membrane potential (Vm)?

Ion fluxes through channels directly influence Vm.

External Ca2+ and cytosolic free [Ca2+]cyt differ by 3-4 orders of magnitude, causing a steep gradient for Ca2+ influx and upon activation (opening) of the channels.

Obviously, to control Vm the net Ca2+ uptake has to be electrically counterbalanced. Vm in

growing root hairs is hyperpolarized and can be more negative than −200 mV. At such

negative Vm, the driving force for the major cation (K+) is inwardly directed and for the major

anion (Cl-) outwardly directed, implying that Vm would depolarize rather than hyperpolarize on

channel opening. Therefore, the plasma membrane-bound proton pump or H+-ATPase is the

only transporter that can electrically counterbalance the depolarizing Ca2+ current (ICa) In the case of an ion, it will move most quickly down a

concentration gradient as well as down a gradient towards opposite charge. (Active transport proteins move ions so that the electric charge is unequally distributed on the two sides of the membrane. This causes what is called a membrane potential and allows passive transport to occur.)

Membrane potential. The inside of the cell is negatively charged in relation to the outside so

the membrane potential is given an electrical value like -60 mV (range = -50 to -200 mV)

Positive ions therefore tend to enter the membrane readily. Negative ions tend to stay out because of attraction to the external

positive charge and from being repelled by the internal negative charge.

How ion channels work Gating: The conformational change between open, the

conformation in which channel allow ions to pass, and closed, in which channel forbid ions to pass.

Ion channels can be classified by their gating mechanism Facilitated Diffusion:

Transmembrane proteins form a channel through the cell membrane Ions pass down a concentration gradient The channel can be opened or closed Does not require energy (ATP)

Active ion pumps:

Moves ions against a concentration gradient

Requires ATP (Active process)

Ions are captured and ejected on the other side of the membrane

Types Of Ion Channels Voltage-Gated: Open or close in response to

change in charge across the plasma membrane. They are found in muscle, neuron and plant cells.

Ligand-Gated: Open or close in response to binding of small signaling molecule (ligand). Ligand can be intra cellular or extra cellular.

Stretch-activated: They are mechanically gated channels, and opening of these channels creates nerve impulses. In plants: in touch responses.

http://www.omedon.co.uk/ionchan/channels

Opening of ion channels that are either an integral part of the receptor molecule (ligand-gated ion channels) or that are linked to the receptor through a G-protein mediated mechanism (ion channel-linked receptors). depolarizes the membrane

In contrast, voltage-gated channels open or close in response to a change in voltage across the cell membrane.

66

Proteases in signal transduction

• Signaling by proteolysis. The anthrax toxin is composed of three proteins. One of these is a metalloproteinase termed lethal factor (LF). The cellular target of LF is a MAPKK. Cleavage of MAPKK results in an impairment of the MAPK cascade Also, in plants

• Yersinia pestis is the infectious agent responsible for black death. One of the effectors delivered from the bacteria to the cytosol of the target cell is YopJ, a 33-kD protein that inhibits a number of signaling pathways. YopJ is a cysteine proteinase that can remove SUMO (small ubiquitin-like modifier) residues from proteins, resulting in alteration of signaling pathways (7). Also, in plants

• Regulated intramembrane proteolysis (RIP). In RIP, a transmembrane protein is cleaved within the membrane that results in release of a fragment into the cytosol, which translocates to the nucleus to activate gene transcription. Examples of RIP include the Notch signaling pathway Also, in plants

67

END of Part I

Part IIForward/Reverse Genetics

of

Environmental stress signaling

68

Signaling strategies

• Molecular biology succeeded in reducing the functions of individual proteins to chemistry, and discovered the principles that govern the transmission and expression of genetic information

• But how do different molecules form a signaling network, and how do these networks interact to allow cells to mount appropriate responses to an enormous number of different combinations of stimuli?

• How do networks arise and evolve?

Evolution seems has maintained only a fraction of possible networks

• A linear cascade generally reflects a moving average of its input

• Many signaling networks have both positive- and negative-

feedback loops. They can give rise to a variety of behaviors,

including switches that remain in their active state after the

stimulus has been removed, oscillators, etc.