Cellular Signal Transduction

Signal transduction is the process by which an extracellular signaling molecule activates a membrane receptor, that in turn alters intracellular molecules creating a response. There are two stages in this process: a signalling molecule activates a certain receptor on the cell membrane, causing a second messenger to continue the signal into the cell and elicit a physiological response. In either step, the signal can be amplified, meaning that one signalling molecule can cause many responses.

Signal Transduction and gene regulation in plant development and stress responses

Signaling molecule Receptor of target cell Signal transduction Intracellular molecule

biological effect

Pathway

Signaling molecules
Signaling molecules, which are released by signal-producing cells, reach and transfer biological signals to their target cells to initiate specific cellular responses.

Receptor
Receptors are specific membrane proteins, which are able to recognize and bind to corresponding ligand molecules, become activated, and transduce signal to next signaling molecules. Glycoprotein or Lipoprotein

ligand
A small molecule that binds specifically to a larger one; for example, a hormone is the ligand for its specific protein receptor.

 Membrane receptors
membrane

Glycoprotein
Intracellular receptors

Cytosol or nuclei
DNA binding protein

Signalling molecules
Signal transduction involves the binding of extracellular signalling molecules and ligands to cell-surface receptors that trigger events inside the cell. Intracellular signaling cascades can be started through cell-substratum interactions. Receptors- membrane proteins, membrane potential,proteinaceous pores,channels C- terminal region of transmembrane protein receptor is phosphorylated by protein kinases

Proteins and peptides: Hormones, cytokines Effect by membrane Amino acid derivatives: receptors Catecholamines Fatty acid derivatives: Extracellular molecules Prostaglandins Signal molecules Intracellular molecules Effect by intracellular receptors
Steroid hormones, Thyroxine, VD3

cAMP, cGMP, IP3, DG, Ca2+

Transduction of abiotic signals to altered gene expression at the cellular level

Ozone,extreme temperatures,flooding, drought,salt

Plants
Physiological & developmental events

Sress recognition Signal transductionAltered cellular metabolism

Gene expression patterns often change in response to stress:

Changes in metabolism & development leads to altered gene pattern These changes are integrated into a response by the whole plant that may modify growth ,development and even influence reproductive capabilities Clues from yeast & bacterial proteins show that these proteins initiate signal transduction in response to abiotic stress (e.g. low osmotic potential) ,and also involves hormones (ABA,JA,ethylene) & secondary messengers (Ca 2+)

Model depicting various possible events involved in abiotic stresses

(1)stress perceival, (2) stress signal transduction (3) transcriptional activation of stress genes (4)synthesis and accumulation of stress proteins, resulting finally in (5) biochemical, (6) cellular and (7) Physiological manifestations

Elements of signal transduction

Intracellular Ca++(Second messenger)information from extracellular source to target with in the cell Protein kinases (enzyme that phosphorylate & alter the activity of target protein)-

serine (Ser)
H H3N+ C CH2 OH COO

threonine (Thr)
H H3N+ C COO

CH OH CH3

Many enzymes are regulated by covalent attachment of phosphate, in ester linkage, to the side-chain hydroxyl group of a particular amino acid residue (serine, threonine, or tyrosine).

Protein Kinase
Protein OH + ATP Protein O

O P O O + ADP

Pi

H2O

Protein Phosphatase

A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on a protein.
A protein phosphatase catalyzes removal of the Pi by hydrolysis.

Phosphorylation may directly alter activity of an enzyme, e.g., by promoting a conformational change. Alternatively, altered activity may result from binding another protein that specifically recognizes a phosphorylated domain. E.g., 14-3-3 proteins bind to domains that include phosphorylated Ser or Thr in the sequence RXXX[pS/pT]XP, where X can be different amino acids. Binding to 14-3-3 is a mechanism by which some proteins (e.g., transcription factors) may be retained in the cytosol, & prevented from entering the nucleus.

Protein Kinase
Protein OH + ATP Protein O

O P O O + ADP

Pi

H2O

Protein Phosphatase

Protein kinases and phosphatases are themselves regulated by complex signal cascades. For example:
Some protein kinases are activated by Ca++calmodulin. Protein Kinase A is activated by cyclic-AMP (cAMP).

Protein Kinase A (cAMP-Dependent Protein Kinase) transfers Pi from ATP to OH of a Ser or Thr in a particular 5-amino acid sequence. Protein Kinase A in the resting state is a complex of: 2 catalytic subunits (C) 2 regulatory subunits (R). R2C2

R2C2
Each regulatory subunit (R) of Protein Kinase A contains a pseudosubstrate sequence, like the substrate domain of a target protein but with Ala substituting for the Ser/Thr. The pseudosubstrate domain of (R), which lacks a hydroxyl that can be phosphorylated, binds to the active site of (C), blocking its activity.

R2C2 + 4 cAMP R2cAMP4 + 2 C

When each (R) binds 2 cAMP, a conformational change causes (R) to release (C).
The catalytic subunits can then catalyze phosphorylation of Ser or Thr on target proteins. PKIs, Protein Kinase Inhibitors, modulate activity of the catalytic subunits (C).

Phosphodiesterase enzymes catalyze:

cAMP
N

NH2 N

cAMP + H2O AMP

The phosphodiesterase that cleaves cAMP is activated by phosphorylation catalyzed by Protein Kinase A.
O

N H2 5' C 4' O H H

1'

Thus cAMP stimulates its own degradation, leading to rapid turnoff of a cAMP signal.

H 3' P O O-

2' H

OH

Signal amplification is an important feature of signal cascades: One hormone molecule can lead to formation of many cAMP molecules. Each catalytic subunit of Protein Kinase A catalyzes phosphorylation of many proteins during the lifetime of the cAMP.

Phosphatidylinositol Signal Cascades

O O R1 C O H2 C CH H2 C O O C O P O OH
2 1

R2

O H
6

OH
5

H OH
3

OH H H
4

phosphatidylinositol

OH

Some hormones activate a signal cascade based on the membrane lipid phosphatidylinositol.

O O R1 C O H2C CH H2C O O C O P O OH
2 1

R2

O H
6

OPO32
5

H OH
3

OH H
4

H PIP2 phosphatidylinositol4,5-bisphosphate

OPO32

Kinases sequentially catalyze transfer of Pi from ATP to OH groups at positions 5 & 4 of the inositol ring, to yield phosphatidylinositol-4,5-bisphosphate (PIP2). PIP2 is cleaved by the enzyme Phospholipase C.

Different isoforms of Phospholipase C have different regulatory domains, & thus respond to different signals. A G-protein, Gq activates one form of Phospholipase C.

O R1 C O

H2C CH H2C

C O

R2

P O OH
2

O H
1 6

cleavage by Phospholipase C

OPO32
5

H OH
3

OH H
4

H PIP2 phosphatidylinositol4,5-bisphosphate

H OPO32

When a particular GPCR (receptor) is activated, GTP exchanges for GDP. Gqa-GTP activates Phospholipase C. Ca++, which is required for activity of Phospholipase C, interacts with () charged residues & with Pi moieties of the phosphorylated inositol at the active site.

OPO32 H OH
2 1 6

OPO32
5

O O R1 C O H2C CH H2C OH O C R2

H OH
3 4

OH H

H OPO32

IP3 inositol-1,4,5-trisphosphate

diacylglycerol

Cleavage of PIP2, catalyzed by Phospholipase C, yields 2 second messengers: inositol-1,4,5-trisphosphate (IP3) diacylglycerol (DG). Diacylglycerol, with Ca++, activates Protein Kinase C, which catalyzes phosphorylation of several cellular proteins, altering their activity.

Ca++ IP3 Ca

calmodulin

Ca++-release channel
++

endoplasmic reticulum

ATP

Ca++-ATPase ++ ADP + Pi Ca

IP3 activates Ca++-release channels in ER membranes. Ca++ stored in the ER is released to the cytosol, where it may bind calmodulin, or help activate Protein Kinase C. Signal turn-off includes removal of Ca++ from the cytosol via Ca++-ATPase pumps, & degradation of IP3.

OPO32 H OH H OH H H OPO32 OH H H OPO32 OH

OH H OH H H

H OH OH H H OH

(3 steps)

+ 3 Pi

IP3

inositol

Sequential dephosphorylation of IP3 by enzyme-catalyzed hydrolysis yields inositol, a substrate for synthesis of PI. IP3 may instead be phosphorylated via specific kinases, to IP4, IP5 or IP6. Some of these have signal roles. E.g., the IP4 inositol-1,3,4,5-tetraphosphate in some cells stimulates Ca++ entry, perhaps by activating plasma membrane Ca++ channels.

O O R1 C O H2 C CH H2 C O O C O P O O H
1 6

R2

phosphatidylinositol3-phosphate

OH
2

OH
5

OH H OPO32 H
3 4

OH

The kinases that convert PI (phosphatidylinositol) to PIP2 (PI-4,5-P2) transfer Pi from ATP to OH at positions 4 & 5 of the inositol ring. PI 3-Kinases instead catalyze phosphorylation of phosphatidylinositol at the 3 position of the inositol ring.

O O R1 C O H2 C CH H2 C O O C O P O O H
1 6

R2

PI-3-P, PI-3,4-P2, PI-3,4,5-P3, and PI-4,5-P2 have signaling roles.

phosphatidylinositol3-phosphate

OH
2

OH
5

OH H OPO32 H
3 4

OH

Head-groups of these transiently formed lipids are ligands for particular pleckstrin homology (PH) & FYVE protein domains that bind proteins to membrane surfaces. Other protein domains called MARKS are (+) charged, and their binding to () charged head-groups of lipids like PIP2 is antagonized by Ca++.

Protein Kinase B (also called Akt) becomes activated when it is recruited from the cytosol to the plasma membrane surface by binding to products of PI-3 Kinase, e.g., PI3,4,5-P3. Other kinases at the cytosolic surface of the plasma membrane then catalyze phosphorylation of Protein Kinase B, activating it. Activated Protein Kinase B catalyzes phosphorylation of Ser or Thr residues of many proteins, with diverse effects on metabolism, cell growth, and apoptosis. Downstream metabolic effects of Protein Kinase B include stimulation of glycogen synthesis, stimulation of glycolysis, and inhibition of gluconeogenesis.

Gene regulation in plant growth and development

A variety of external & internal signals modify plant cell metabolism , growth, & development The ability of cell s to respond to these signals is not confined to cells that are still growing and developing Mature cells too, can initiate metabolic responses and can even reinitiate growth and division in response to signal information

Characteristics of Signal transduction in Plants

The stream of signals to which plant cell react is continuous and complex Signal transduction uses a net work of interactions with in the cells, among the cells and through out the plant Plant cells contain two information systems; genetic & epigenetic Different signals affect the transduction network in different ways and at different places, but most modify gene expressson

 The bacterial two component system in which a receptor and an effector interact through phosphorylation of histidine and aspartate residueAutophosphorylation of receptor--Phosphorylation of response regulator--dephosphorylation of response regulator (e.g.-Arabdopsis ethylene receptor-ETR1, &cytokinin sensing CKI1/GCR1) Plant hormones as a receptor---ethylene, ABA,GA,IAA,JA,phytochromes etc.

Types of signal transduction pathway in Plants

Saline stress inhibits sucrose synthesis and promotes accumulation of mannitol

Fructose- 6-Po4

Phosphomannose isomerase
Mannose-6-Po4 Mannose -6 Po4 reductase NADPH----NADP Mannitol-1 po4 Mannitol-1 Po4 phosphatse -Pi Mannitol

Accumulation of pinitol in response to salt stress

Glucose-6 Po4 NAD+ myo- Inositol-1 PO4 synthase myo-Inositol- 1- PO4 -Pi myo-Inositol- 1- PO4-phosphatse myo-inositol S-adenosylmethionine--myo-inositol-6-O-methyltransferase S-adenosylhomocysteine Ononitol Ononitol epimerase Pinitol

SOS signaling pathway for ion homeostasis under salt stress in Arabidopsis.

 Salt stress elicited Ca2+ signals are perceived by SOS3, which activates the protein kinase SOS2. Activated SOS2 phosphorylates SOS1, a plasma membrane Na+ /H+ antiporter, which then transports Na + out of the cytosol. The transcript level of SOS1 is regulated by the SOS3-SOS2 kinase complex. SOS2 also activates the tonoplast Na + /H + antiporter that sequesters Na + into the vacuole. Na entry into the cytosol through the Na + transporter HKT1 may also be restricted by SOS2. ABI1 regulates the gene expression of NHX1, while ABI2 interacts with SOS2 and negatively regulates ion homeostasis either by inhibiting SOS2 kinase activity or the activities of SOS2 targets. Double arrow indicates SOS3-independent and SOS2-dependent pathway.

ABA-dependent and ABAindependent signal transduction.

Environmental factors that increase concentration of ROS

Ozone- UV radiations +SO2,NO,NO2,--------------Stratasphoric/TroposphoricDrought, Senesence, Herbicides,(paraquat dichloride) Wounding, Intense light, --OXIDATIVE STRESS Pathogens, Heat&Cold, Heavy metals, Root nodules-----------------------------------------

Generation and scavenging of superoxide radical and hydrogen peroxide, and hydroxyl radical-induced lipid peroxidation and glutathione peroxidase-mediated lipid (fatty acid) stabilization

Role of osmolytes in plant protection against abiotic stress

Some compatible solutes may serve protective functions in addition to OA called osmoprotector. Glycine betain prevents salt- induced inactivation of Rubisco & destabilization of the oxygen evolving complex of PSII Sorbitol,mannitol,myo-inositol, and proline can also scavenge hydroxyl radicals in addition to OA. Transgenic plants can be used to test the acclimative functions of specific osmolytes

Fatty acids signalling molecules

Protein kinases (PK): primary elements in the signal transduction

These are ubiquitous enzymes and are signal specific Catalyses reversible transfer of-PO4 from ATP to Serine,threonine or tyrosine amino acid chains on target proteins Activity is counter balanced by the specific protein phosphatases Activation of PK has been implicated in response to light,pathogen attack,GR,salt,heat temp.stress,nutrient deprivation etc. Several protein kinases are concerned with regulation of metabolic pathways Hundereds of plant genes fdor different PKs have been identified but atleast 1000 must exist(Tab,:various groups of PKs identified in plants

Kinase cascade involved in biotic stress responses in plants

The sensing of stress signals and their transduction into appropriate responses is crucial for the adaptation and survival of plants. Kinase cascades of the mitogen-activated protein kinase (MAPK) class play a remarkably important role in plant signalling of a variety of abiotic and biotic stresses. MAPK cascade-mediated signalling is an essential step in the establishment of resistance to pathogens. Here, we describe the most recent insights into MAPK-mediated pathogen defence response regulation with a particular focus on the cascades involving MPK3, MPK4 and MPK6. We also discuss the strategies developed by plant pathogens to circumvent, inactivate or even hijack MAPK-mediated defence responses.

PAMP- induced MAPK cascade in the plant defence to the bacterial & fungal pathogens

SALT AND DROUGHT STRESS SIGNAL TRANSDUCTION IN PLANTS

Salt and drought stress signal transduction consists of ionic and osmotic homeostasis signaling pathways, detoxification (i.e., damage control and repair) response pathways, and pathways for growth regulation. The ionic aspect of salt stress is signaled via the SOS pathway where a calciumresponsive SOS3-SOS2 protein kinase complex controls the expression and activity of ion transporters such as SOS1. Osmotic stress activates several protein kinases including mitogen-activated kinases, which may mediate osmotic homeostasis and/or detoxification responses. A number of phospholipid systems are activated by osmotic stress, generating a diverse array of messenger molecules, some of which may function upstream of the osmotic stress activated protein kinases. Abscisic acid biosynthesis is regulated by osmotic stress at multiple steps. Both ABA-dependent and independent osmotic stress signaling first modify constitutively expressed transcription factors, leading to the expression of early response transcriptional activators, which then activate downstream stress tolerance effector genes.

Inputs & Out puts:making sense for drought &salt stress signalling

pathways

Figure 1 Functional demarcation of salt and drought stress signaling pathways. The inputs for ionic and osmotic signaling pathways are ionic (excess NaC) and osmotic (e.g., turgor) changes. The output of ionic and osmotic signaling is cellular and plant homeostasis. Direct input signals for detoxification signaling are derived stresses (i.e., injury), and the signaling output is damage control and repair (e.g., activation of dehydration tolerance genes). Interactions between the homeostasis, growth regulation, and detoxification pathways are indicated

THE SOS REGULATORY PATHWAY FOR ION HOMEOSTASIS AND SALT TOLERANCE
High NaC stress initiates a calcium signal that activates the SOS3SOS2 protein kinase complex, which then stimulates the NaC/HC exchange activity of SOS1 and regulates transcriptionally and posttranscriptionally the expression of some genes. SOS3SOS2 may also stimulate or suppress the activities of other transporters involved in ion homeostasis under salt stress, such as vacuolar HC-ATPases and pyrophosphatases (PPase), vacuolar NaC/HC exchanger (NHX), and plasma membrane KC and NaC transporters.

Figure 2 Regulation of ion (e.g., NaC and KC) homeostasis by the SOS pathway.

PROTEIN KINASE PATHWAYS FOR OSMOTIC STRESS SIGNALING

Figure 3 Activation of protein kinases by hyperosmotic stress. The MAP kinase cascade shown is also activated by other stresses. Currently, the functional significance of the kinase activation is unclear (hence the unknown output). SIPK, SIMK, and ATMPK6 are homologous MAP kinases from tobacco, alfalfa, and Arabidopsis, respectively.

OSMOTIC STRESSACTIVATED PHOSPHOLIPID SIGNALING

Figure 4 Phospholipid signaling under salt stress, drought, cold, or ABA. Osmotic stress,cold, and ABA activate several types of phospholipases that cleave phospholipids to generate lipid messengers (e.g., PA, DAG, and IP3), which regulate stress tolerance partly through modulation of stressresponsive gene expression.FRY1(a 1phosphatase) and 5phosphatasemediated IP3 degradation attenuates the stress gene regulation by helping to control cellular IP3 levels. PLC, phospholipase C; PLD, phospholipase D; PLA2, phospholipase A2; PA, phosphatidic acid; DAG, diacyglycerol.

Stress- and ABA-Regulated Gene Expression

Figure 5 ABA metabolism is regulated by osmotic stress at multiple steps. The ABA biosynthesis genes ZEP, NCED, LOS5/ABA3, and AAO are upregulated by salt and drought stresses. ABA degradation is also important in controlling cellular ABA content, and biochemical evidence suggests osmotic stress inhibition of the first step of catabolism

ABA-Dependent and ABAIndependent Signaling

Figure 6 Model showing osmotic stress regulation of earlyresponse and delayedresponse genes. (A) Model integrating stress sensing, activation of phospholipid signaling and MAP kinase cascade, and transcription cascade leading to the expression of delayed-response genes. (B) Examples of early-response genes encoding inducible transcription activators and their downstream delayed-response genes encoding stress tolerance effector proteins. Question marks denote unknown transcription factors that activate the early-response genes.

Fatty acid - derived signals in plants

e.g. Jasmonates Its mutant opr3 in biosynthetic pathway, allow the dissection of cyclopentanone & cyclopentenone signalling In addition, keto,hydroxy & hyperhydroxy FA involved in cell death & stress related gene expression. Bruchins & volicitin as a signal molecule from insects show FA 0derived signalling in plant defence