1. AGR2451 - Lecture 4 (M. Raizada) Notes: -questionnaire and hand-out at the front -this week’s reading on plant hormones on reserve: Page 545-563 in Biology of Plants -Review of previous 2 lectures: 1. How an enzyme works - the active site 2. How amino acids build a 3-D, folded enzyme 3. Biological switches- changes in conformation of a protein, protein-protein-DNA interactions of transcription factors 4. The effect of small charged molecules (Phosphate) on protein activity. 5. DNA -- mRNA--- protein. Why?? 6. Transcription, splicing, mRNA export, translation, post-translational modifications, protein folding,compartment export/import. 7. Life is an orchestra of subsets of genes switching on and off to create diverse cell types/diverse responses.

2. Lecture 4 -Extracellular & Intracellular Signalling Networks I. Coordinating gene expression with the environment To create life, biochemistry had to be placed inside a compartment sequestered from the environment. However, an organism must sense and response to its environment to survive. In particular, a plant is immobile and must respond. The environment is outside of a cell. A eukaryotic gene is inside the nucleus. How does the extracellular environment turn a gene on and off? (class) For Example: cold, pathogen attack, light quality and intensity A) The signal must be perceived at the plasma membrane/wall surface. B) The signal must be transmitted across the plasma membrane. C) The signal must be communicated across the cytoplasm to the nucleus. D) In the nucleus, the signal must interact with the appropriate transcription factors to turn genes on and off. “Signal Transduction Cascade” =a series of molecular switches to communicate from the cell surface to a group of genes From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.977 ASPP, Rockville MD, 2000 Slide 4.2

3. Signal Transduction Cascade A) The signal must be perceived at the plasma membrance/wall surface. B) The signal must be transduced across the plasma membrane. How? A stimulus causes a change in conformation of a protein receptor embedded in the plasma membrane. A molecular stimulus is called a ligand. The receptor consists of an extracellular (sensing) domain, a membrane spanning domain that is hydrophobic, and an intracellular domain that responds to the change in conformation to signal other proteins/enzymes: Slide 4.3A From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.937 ASPP, Rockville MD, 2000 The surface of a pathogen or a molecule released from a pathogen (cell wall fragment) can act to turn on a plant receptor to activate the plant disease-resistance/defence response. Hence, plant receptors are crucial for survival and a key for crop improvement. C) A common way to communicate the signal from the membrane to the nucleus is for the activated receptor to transfer a phosphate molecule onto another enzyme thereby activating it (the phosphate changes its conformation into a functional state). This enzyme is not anchored to the membrane but diffusible inside the cytoplasm. It is called a secondary messenger. What is a kinase? an enzyme that transfers a phosphate, eg. to another enzyme. There are at least 340 kinase proteins in Arabidopsis.

4. Slide 4.3B Signal Transduction Cascade C) Example of a receptor kinase adding a phosphate to activate a cytoplasmic secondary messenger From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.943 ASPP, Rockville MD, 2000 One kinase can activate a second kinase which can activate a third kinase, etc. Why did evolution build a cascade of second messengers in the cytoplasm rather than a single secondary messenger from the plasma membrane to the nucleus? 1. Amplify signal. 2. Many more control points.

5. Signal Transduction Cascade Slide 4.3C From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.962 ASPP, Rockville MD, 2000 D) The signal must interact with the appropriate transcription factors to turn genes on and off. How? The secondary messengers must travel through the nuclear pore into the nucleus, and there they directly bind to specific transcription factors and/or add a phosphate to the transcription factors thereby changing their conformation, causing them to be activated or repressed (causing whole sets of genes to be transcriptionally turned on or off). Those genes that commonly share the DNA sequence (promoter or enhancer) to which the activated transcription factor binds become coordinately regulated.

6. A cell must be able to coordinate multiple environmental inputs. How is this achieved? Slide 4.4A Growth gene 6-20 base DNA enhancer sequences Light- Nitrogen- Drought- Responsive Coordinating Multiple Environmental Inputs A) Combinatorial control of gene regulatory regions. What is this and how is it achieved? One gene may have a regulatory region consisting of multiple enhancer sequences each of which binds a different transcription factors, from a using a unique signal transduction cascade. From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.933 ASPP, Rockville MD, 2000

7. B) Cross-talk between signal transduction cascades. The different signalling pathways may enhance or interfere with one another by binding to one another’s enzymes or transferring or removing phosphate molecules. An enzyme that removes a phosphate = phosphatase. There are at least 70 different phosphatases in Arabidopsis. From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.984 ASPP, Rockville MD, 2000 Coordinating Multiple Environmental Inputs Why is such cross-talk between signal transduction enzymes useful to the cell? Can respond to a stimulus (eg. Pathogen attack) by turning on/off different responses in a coordinated way. Slide 4.4B

8. II. Cell-to-cell local coordination B) What are plasmodesmata? They are direct cytoplasmic connections between adjacent cells. This allows small molecules such as Ca++ and even transcription factors to move between adjacent cells in a very regulated manner. From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.751and p.1021 ASPP, Rockville MD, 2000 Slide 4.5 When multicellular organisms developed, evolution needed to allow cells to send information to neighboring cells to give them positional information. A) Cell-cell receptor-ligands. An adjacent cell exports a small protein (peptide) or a chemical to its cell surface which binds to the receptor of an adjacent cell to turn on/off a specific set of genes or activate/repress specific enzymes.

9. AGR2451 - Lecture 6 - October 2nd III. Long-distance signalling As multicellular organisms became very complex, with distinct organs, then gene expression in distant cells needed to be coordinated in order for the organism to develop properly and to respond to the environment. A) This can be mediated by a wave of diffusible Ca++ ions. These ions then bind to other enzymes to activate/repress them. Similar to phosphate, Ca++ can also be used as a messenger inside a cell: in this case, the Ca++ may be released from the vacuole where it is stored, into the cytoplasm. Slide 4.6 Cold-induced wave of Ca++ in a tobacco leaf. From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.972 ASPP, Rockville MD, 2000

10. III. Long-distance signalling B) The main method, however, are hormones, long-distance signalling molecules. C) Each cell may respond to the same signal in a different way (for example: in drought, the leaves may stop expanding to decrease evaporation while the roots might extend to find water). Hence, there must be cell-specific receptors or secondary messengers. Slide 4.7A From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.851 ASPP, Rockville MD, 2000

11. III. Long-distance signalling - The Major Plant Hormones Slide 4.7B HormoneSynthesized where?Function/Notes Auxinleaf primordiaapical dominance young leavesroot induction vascular tissue development stimulates fruit development many others Cytokininsroot tipscell division shoot formation Ethyleneripening or fruit ripening senescence tissuestissue senescence Abscisic acidmature leaves, stomatal closure especially afterembryogenesis water stressinduces sugar transport from leaves to seeds induces storage protein synthesis in seeds Gibberellinsyoung shoots and elongation of shoots* developing seeds seed germination stimulates flowering *Why was a mutation that altered Gibberellin synthesis (allele Norin10) enormously responsible for the Green Revolution? Shortened plant height in response to added fertilizer, preventing plants from falling over and losing seeds.

12. Hormones - Example Ethylene Ethylene is a diffusible gas. One of its functions is to promote fruit ripening. Ripening fruit releases this gas (hence this is why one bad apple spoils the whole bag). Cell wall degrading enzymes Pigment activation Slide 6.7C Ethylene = C 2 H 4 Ethylene binds to a receptor and the signal is transmitted via a phsphorylation kinase cascade to the nucleus where the signal activates transcription factors which turn on genes involved in fruit ripening such as cell wall degrading enzymes, pigment activation and sugar release. From Biochemistry and Molecular Biology of Plants (W.Gruissem, B. Buchanan and R.Jones p.980 and 1075 ASPP, Rockville MD, 2000

13. Protein-Protein Interactions are Complex Slide 6.9 Biologists are slowly determining which proteins interact with other proteins for signalling, for biochemical pathways, and to create higher order structures (such as microtubule cables). The result is the creation of protein-interaction maps that display entire networks of proteins working together. These maps illustrate that: A) many proteins are required for every process, not single proteins. B) It illustrates that signals from the environment play a major role in the molecular events inside a cell. C) In the future, such maps will be at the forefront of advances in both agriculture and medicine. Eisenberg et al. (2000) Nature 405, 823-826 Nature Publishing Company, UK. Example of a Yeast protein Interaction map Slide 4.8 Can plan new herbicides and pesticides

14. IV. Evolution by altering “Master Switches” What is a "Master Switch"?? It is a single molecule (Transcription factor, receptor, messenger) which switches on/off large numbers of genes or enzymes. For example, there is a Master Switch to make eyes, switches to make flowers, etc. Changes in the intensity of the switch or when and where the switch acts can thus profoundly alter how an organism interacts with its environment or how it develops. These small changes have likely led to new species and dramatic changes during evolution. Similarly, changes in transcription factors have also resulted in large changes in phenotype with only a small change in genotype. V. Summary of Key Concepts Biology has protein-based switches, including transcription factors (on/off) and changes in protein conformation by Calcium/Phosphate The environment interacts with an organism by affecting one or more of these switches. The environment turns genes on/off using a plasma membrane receptor that activates a signal transduction cascade ultimately resulting in the switching on/off of gene expression. There are also receptors and molecules that allow adjacent cells and distant cells to communicate. Hormones allow an organism to have a coordinated response,though different cells may turn on/off different genes in response to the same hormone. A single gene may be turned on/off by multiple environmental stimuli (combinatorial control) and different signalling cascades may interfere with one another. Slide 4.9