Life on the edge or

1. Life on the edge or As sesile organisms plants must live wherever they happen to germinate And they usually manage Salt lake

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

3. Urggh!! Irritability-Sensitivity Irritability is one of the major defining characteristics of all living organisms – the ability to respond to the environment 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 Biotech is a pioneer in the application of functional genomicsin plants. Mendel's mission is to discover and characterize the function of plant regulatory genes and to develop high-value products 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.

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 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 irrelevant Detecting signals at low concentrations Translating diverse signals into a 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 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 concentrations Saturability – 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 Solutions Sorting out the [ir]-relevant signals Receptors with a high degree of specificity Detecting signals at low concentrations Receptors with high affinity coupled to an amplification Activation of signalling pathways designed around a limited number of common processes

13. Signalling 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…. 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. TF Nucleus TF Steroid hormone GR However the majority of signalling systems aren’t that simple

14. Signal transduction in plants http://fig.cox.miami.edu/~cmallery/150/memb

15. Coordination of stress responses • 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

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 • Plasma membrane, • tonoplast, • 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. 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

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 External stimulus can trigger Ca2+ influx from INTERNAL stores Conformational changes in IP3R transduce subsequentsignaling. The distribution of positive and negative charges stabilizes the open conformation of the channel and allows the entry of Ca2+into the cytoplasm.

26. Principles of information transmission Environmental input The Goal of signaling pathways is to produce appropriate reactions to the type &strength of stimulus processing reaction growth movement proliferation EFFECTOR FUNCTIONS death Generate new information distribution of information by 2nd messengers necrosis apoptosis

27. RECEPTOR SUBTYPES Getting 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 Plasma membrane outside I. 7tm • Seven-transmembrane (serpentine) • Ligands include proteins, peptides, • lipids, other small molecules • Signal via heterotrimericG-proteins • and second messengers • Ligand binding initiates signal • via G-protein activation membrane receptors inside interact with Transmembrane domain III. Ion channel receptors Intracellular domain – couples to next step (may have enzymatic activity) • Multi-transmembrane • >20 known • Signal via conduction of ions across • membranes • Ligand binding initiates channel opening II. RTKs

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. On-Off Switches – GTP-Binding Proteins 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. Exchange of bound GDP for GTP g b a GTP GDP GDP INACTIVE a a subunit dissociates from bg GTP ACTIVE Pi a a subunit GTPase activity GTP>GDP+Pi GDP Active a subunit can interact with and activate the next step in the signalling pathway a subunit reassociates with bg

32. p21ras p21ras p21ras GDP GDP GTP On-Off Switches – GTP-Binding Proteins 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 This causes exchange of bound GDP for GTP p21ras Guanine nucleotide exchange factor interacts with ras Activated ras interacts with and activates the next component in the signalling pathway On GTP GDP ACTIVE INACTIVE Pi Ras GTPase stimulated by association with GTPase-activating protein (GAP) Off Intrinsic GTPase activity hydrolyses GTP to GDP and Pi

33. Most Small GTPases Are Highly Conserved Regulators of Intracellular Trafficking

34. Crosstalk between GPCRs and small GTPases 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. A) The binding of agonist to a GPCR promotes GTP exchange on the Ga subunit, which leads to its dissociation from Gbg subunits. The activated proteins Ga and Gbg 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 Gbg) 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

35. Activation of NADPH Oxidase by a small GTPase Rac1 dominant dominant

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

37. O- O- C C O O C C O O H H C C C C O O P P O O C C C C O O Serine Serine NH NH O- O- NH NH On-Off Switches – Protein Phosphorylation Protein Kinases– transfer phosphate from ATP to specific amino acids Protein Phosphatases– remove phosphate from specific amino acids Kinase Phosphatase ATP ADP Pi Phosphorylation Dephosphorylation O-Phosphoserine

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 EGFR Ras is membrane-anchored by a farnesyl moiety Sos Ras GDP Pro Pro Pro Pro Pro Ras is now accessible to Sos SH3 SH2 Ras SH3 GTP GDP

42. Signaling from Ras GTPases • Receive Ras signal at • membrane • Ser-directed • Activated by Ser • phosphorylation • Thr/Tyr-directed • 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 MAPKKK* MAPKK MAPKKK* • Activated by Thr/Tyr • phosphorylation • Ser-directed • Can translocate to • nucleus MAPKK MAPK MAPK Enter nucleus to phosphorylate transcription factor (the TARGET) MAP kinases (ERKs, JNK, p38)

43. History: cloning the first MAPK Gene (MPK4) 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. (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

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 The Plant life cycle dormancy  germination  growth and development  flowering and fruiting  death http://aggie-horticulture.tamu.edu/faculty/davies/students/ngo

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 The Biochemical approach Ca2+ ? Stomatal closure (K+ efflux, turgor decrease) ABA ROS ABA ↓ 5 min Stomatal pore

48. The Biochemical approach The effect of H2O2 and Ca2+ on stomatal opening. Measurements of Ca2+ currents in guard cells

49. The Biochemical approach ABA activates Ca2+ channels via NADPH oxidase In absence of NADPH, ABA does not induce Ca2+ influx, (thus ROS production is generated prior to Ca2+ increase).

50. Genetic approach Every activity is executed by a protein (or RNA) that is encoded by a gene ABA does not activate Ca2+ channels in abi mutants. H202 activates Ca2+ channels in abi1-1 but not in abi2-1