COURSE OBJECTIVES: to gain knowledge about - plant responses to environmental stress (physiological, biochemical, genetic) - research approaches for study of environmental stresses. biochemical, genetic and molecular on one hand mechanisms responsible for environmental stress tolerance on the other hand the factors causing injury during stress. integrate concepts from related disciplines To sharpen written and oral communication skills associated with research

Detailed outline of the Plant Stress course 72637 - גנטיקה מולקולרית של תגובות צמחים לעקות סביבתיות ( ד"ר אלכס לוין חדר 2-468, טל: 86543 (שעות קבלה יום ב' מ-14:00 Plant interactions with surrounding environment Plasma membrane as the barrier and stress sensor Oxygen and Reactive Oxygen Species in environmental stress response (ROS chemistry, reactions, antioxidants, signaling) Free radicals & antioxidants: Nitric oxide, oxidative stress, hypoxia Stress signal transduction pathways: Biochemical vs. genetic analysis and the novel genomics/functional/ comparative genomics approaches Major regulatory signaling molecules (stress hormones, ABA, ethylene) Intracellular messengers: a) Ca2+ as a general and specific signaling molecule b) Phospholipids in regulating stress signal transduction chapter 18 (Trawewas:Signal Perception and Transduction) Buchanan et al, eds. (2000). Biochemistry &Molecular Biology of Plants http://www.aspb.org/downloads/BiotextCH18.pdf Molecular genetics of Specific stresses: a) salt stress b) other stresses (temperature/UV/ozone/polution) Genetic engineering of abiotic stress resistance

Environmental stimuli that affect plant growth Plant response to environmental stimuli involves perception, transduction, adaptation Sensing changes in the surrounding environment Responding to gravity and direction of light, etc. Adjusting their growth pattern and development Control systems in plants involve adaptations, adaptations, adaptations To adapt and to optimize the growth plant needs to monitor everything: bio, envir, endogenous Plants need to monitor everything in order to optimize growth (i.e. to adapt) to environmental conditions, endogenous present & future

Plants have to exploit their immediate environment to maximum effect Plants have to exploit their immediate environment to maximum effect. Their inability to move means that the best way of dealing with stress is by physiological or morphological changes. Abiotic stresses, and ways to adapt to them are numerous and interlinked there’s more than one way to skin a cat

Abiotic Water Oxygen Nutrients Temperature Salt stress Pollutants excess or deficit Biotic Insects Weeds Pathogens Plant competition mutations In biology, stress is the driving force behind the process of adaptation and evolution

Resistance to drought and salt stresses is interlinked

Example of elucidating stress responses כל חץ משמעו מנגנון SIGNAL TRANSDUCTION Inputs for ionic and osmotic signaling pathways are ionic (excess Na+) and osmotic (turgor) changes. The output of ionic and osmotic signaling is cellular and plant homeostasis. Annual Review of Plant Biology 53: 247

Na+ UPTAKE/EXTRUSION IN THE PLANT CELL Plasma Membrane PPi H+ Na+ Na+ K+ H+ High-affinity K+ transporters V-PPase Na+ H+ Na+/H+ antiport Vacuole Na+ Na+ Tonoplast V-ATPase K+ Primary active P-ATPase uses the energy of ATP hydrolysis to pump H+ out of the cell generating an electrochemical H+ gradient. This generated proton motive force operates the secondary active Na+/H+ antiport which extrudes Na+ against its electrochemical gradient coupled with the movement of H+ into the cell along its electrochemical gradient. The primary active V-ATPase and V-PPase (pyrophosphatase) energize the tonoplast for secondary active transport of Na+ into the vacuole by the Na+/H+ antiport. Plant cells lack Na+/K+ ATPase Ion ratios are altered by influx of Na+ thus plants maintain low cystolic Na+ [] and high cystolic K+/Na+ ratio Na+ [] is maintained in both halophytes and glycophytes at non-toxic levels by compartmentalization of Na+ into vacuoles which averts the deleterious affects of Na+ in the cytosol. Na+ can also enter via KUP/HAK/KT K+ transporters, cyclic-nucleotide-gated channels, glutamate-activated channels, LCT transporters and HKT transporters K+/Na+ selectiveVICs H+ ATP ATP K+/Na+ ratio H+ P-ATPase Adapted from Mansour et al. 2003

ASPECTS OF SALT TOLERANCE IN PLANTS Ionic stress Ion homeostasis SOS3 SOS2 Ion transporters SALT STRESS Homeostasis Osmotic stress MAPK cascade ? Osmotic homeostasis Osmolytes COLD DROUGHT ABA Detoxification Secondary stresses Stress proteins CBF/DREB Cell division and expansion Adapted from J-K Zhu 2001

Discovery vs. Hypothesis-Driven Science scientific projects test a hypothesis or a model that arises during analysis of experimental data. Hypothesis is formulated and experiments support or refute the hypothesis. While this approach is valid, it can preclude discovery of variables, and mechanisms that are unknown during the study design. An alternate approach is to nonselectively gather information about a particular biological system; the results are then analyzed with the hope that significant characteristics will emerge, providing insight into the mechanism. This approach is known as discovery science. Discovery science and hypothesis-driven science are complementary approaches. Discovery science is a high throughput method that can screen fast many genes for potential involvement in a biological process. in contrast, Hypothesis-driven science is low throughput method

Discovery and Hypothesis approaches to Science The Genome Projects are the first modern practice of discovery science, now followed by other omics The objective of discovery science is to define all elements in a system and to create a database containing that information. For example, discovery approaches provide the complete sequences & expression of the organism genes. The transcriptomes and proteomes of all cell types Discovery science lies in contrast to hypothesis-driven science, which creates hypotheses and attempts to distinguish among them experimentally. Integration of the discovery and hypothesis approaches is area of systems biology.

The Four Elements of Abiotic Stress Water Light Nutrients Temperature time stress Plant stress can be defined in many ways - primarily, it is induced by any environmental factor, which is capable of producing or inducing a potentially harmful shift in the ability of a plant to exist under its ideal conditions. In other words, stress may be induced by the excessive pressure of a factor, on combination of factors (which may be abiotic or biotic) which it turn expresses an effect on the normal functioning of a process or series of processes within plants. Stress may be short-term, in which case, there will be recovery of normal cellular and biochemical processes within a reasonable time frame, or they may be long term, in which case, the effects that manifest themselves as a result of the perturbation, may have more serious and deleterious consequences. The term "stress" when used in plant biology, should be used only to refer to the physiological state induced by injurious strain, or inhibition of functioning (Cowan, 1994). in general, performance below optimal genetic potential is indicative of stress

Plant Responses to Stress Mechanical concept of stress Stress is a force per unit area Strain is a change in dimension in response to stress (in other words, deformation of a physical body under the action of applied forces) Failure of a material occurs when the material cannot strain sufficiently to resist stress

Plant Responses to Stress Biological concept of stress Abiotic (physical or chemical) or biotic factor adversely affecting an organism Measured as effect on growth rate and productivity

  average losses  Crop  record yield*  average yield*  disease  insect  weed  other (abiotic)  corn  19,300  4,600  750  691  511  12,700  wheat  14,500  1,880  336  134  256  11,900  soybean  7,390  1,610  269  67  330  5,120  sorghum  20,000  2,830  314  423  16,200  oats  10,600  1,720  465  107  352  7,960  barley  11,400  2,050  377  108  280  8,590  potatoes  94,100  28,300  8,000  5,900  875  50,900  sugar beets  121,000  42,600  6,700  3,700  61,300 % of record yield  21.6%  4.1%  2.6%  69.1% A comparison of the record yields and the average yields indicates that mostly crops are only reaching 20% of their genetic potential due to biotic categories: disease, insect and weeds. The major reduction in yield (~ 70%) is due to abiotic stress. The most significant abiotic stress is water stress, both deficit stress (drought) and excess stress (flooding, anoxia).

Factors that determine plant stress responses

Strategies of stress tolerance in plants Susceptibility -slowed growth--senescence--death Avoidance -deep rooting -short life cycle -leaf modifications Resistance -ex. can survive desiccation of protoplasm “resurrection plants” constitutive deep roots constitutive succulent Example of const overexpression of a cold stress resist protein DREB1A (freeze tolerance) is a dwarf If plants can induce stress resisting genes  Why these genes are not constitutively on? induced freezing toleranceresistance Drought avoidance

I. Important concepts of stress physiology Stress– external factor that is disadvantageous to plants; survival, growth, development, yield Acclimated (Hardened)- increased stress tolerance as a result of prior exposure to a stress condition Cross Resistance- tolerance to a stress based on exposure to a previous stress event of a different nature Adaptation- is a genetically determined level of resistance acquired by a process of selection over many generations

Assignment 1 Define what are the hottest topics in plant research today Start with general definitions, and identify the details Define what is already known Relevance to other systems and/or applicative aspects

Responses to Environmental Stresses 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 provide bacteria with the ability to tolerate randomly fluctuating environmental variation How many genes are likely to be involved with the specification of signaling components. Signaling, in its broadest sense, is now the major area of plant and environmental sciences research

I. SIGNAL TRANSDUCTION AND PLANT RESPONSES Signal-transduction pathways link cellular responses to environmental stimuli Signal transduction pathway = A mechanism linking a mechanical or chemical stimulus to a cellular response. Three steps: reception, transduction, and induction Many signals interact cooperatively and synergistically with each other to produce the final response: red and blue light, nutrients and light. The membrane potential can act as a receptor.The plasma membrane uses pumps and proteinaceous pores,called channels, to control the flux of ions into and out of cells. Potential open a group of voltage-gated channels that allow Ca 2+to enter and thus activate a transduction cascade.

Three steps: reception, transduction, and induction

about the Signals and Signaling A molecule in a biological system encodes information in its shape, charge, hydrophobicity, and reactivity. Any change in a molecule’s composition encodes new information in the amount, rate, and duration of that change and where in the cell or organism the change occurs So, the signaling information is present in what, where, how much and when note that a signal is a signal only when there is a receptor to decipher the information

Plants respond to stress on a cellular and on the whole plant levels link between biotic and abiotic stress signal transduction and plant development The work reveals an important regulatory role of BONZAI1 (BON1) in connecting temperature-dependent growth control and the repression of signalling of a TIR-NBS-LRR resistance gene, SNC1. The growth defect of bon1-1 is a result of constitutive defense signaling involving EDS1, an EDS1 homologue, PAD4, SID2/ICS and SA in a positive feedback loop, but not NDR1 (required for some CC-NBS-LRR signaling). Wt Arabidopsis plants achieve a similar size when grown at temperatures ranging from 16 to 30°C. The genetic control of this growth homeostasis was revealed by mutants (such as acaulis1, acaulis 3, acaulis4, and bonzai1 [bon1]) that cannot maintain constant size at different temperatures (Akamatsu et al., 1999 ; Hua et al., 2001 ). The loss-of-function mutant bon1-1 has a dwarf phenotype mostly because of reduced cell size at 22°C, but it resembles the wild type at 28°C, indicating an essential role of BON1 in growth homeostasis (Hua et al., 2001 ). BON1 encodes a member of the copine gene family that is highly conserved among protozoa, plants, nematodes, and mammals (Creutz et al., 1998 ). The deduced BON1 protein, like other copine proteins, has at its N terminus two calcium-dependent phospholipid binding C2 domains that are mostly found in signal transduction or membrane trafficking molecules (Rizo and Sudhof, 1998 ). BON1 binds to phospholipids in a calcium-dependent manner in vitro, and it is localized to the plasma membrane in plants (Hua et al., 2001 ). The C-terminal region of BON1 shows homology to the A domain of integrins (Williams et al., 1999 ) and is proposed to mediate protein–protein interaction, to possess an intrinsic protein kinase activity, or both (Caudell et al., 2000 ). The A domain in BON1 mediates the interaction of BON1 with its putative functional partner BAP1 that also contains a C2 domain (Hua et al., 2001 ). The presence of C2 domains in both BON1 and BAP1 suggests that they are involved in a biological process regulated by the membrane system, calcium state, or by both factors. In addition to regulating growth homeostasis, BON1 is also shown to modulate defense responses. A bon1 mutant allele cpn1-1 (which we will refer to as bon1-4) exhibits precocious cell death and enhanced disease resistance under low humidity or low temperature conditions (Jambunathan et al., 2001 ; Jambunathan and McNellis, 2003 ). It is intriguing that both growth and defense are affected by bon1 mutations in a temperature-dependent manner. Emerging molecular data suggest that these two processes are intricately intertwined. Mutations or transgenes that perturb cellular metabolism and plant growth cause lesion mimic phenotypes and defense responses characteristic of systemic acquired resistance (SAR) (Mittler et al., 1995 ; Molina et al., 1999 ; Clough et al., 2000 ). Compromised cell growth is found in some mutants with constitutive pathogen responses, such as cpr1 and mpk4 (Bowling et al., 1994 ; Petersen et al., 2000) bon1 are miniature at 22oC but like wild-type at 28oC Responses to Biotic and Abiotic stresses are connected genetically: growth regulation by BON1 is mediated through defense responses. BON1 is a negative regulator of a Resistance (R) gene SNC1. The bon1-1 loss-of-function mutation activates SNC1, leading to constitutive defense responses and, consequently, reduced cell growth

Plant Response to Stress Plants adapt to changing environmental conditions through changes in expression patterns of numerous genes. There is a group of genes whose expression confers resistance to a given stress. There is a common core of defense genes, which responds to several different stresses (general stress-response genes) versus stress-specific genes. Increase in expression of protective genes is co-regulated and is correlated with resistance to oxidative stress.

Plants are more exposed to toxicity by reactive oxygen species O2 availability/accessability high O2 concentration 3) Multiple sources of O2.- 4) exposure to UV 5) rapid temperature shifts 6) salinity, drought 7) necrotrophic pathogens that secrete O2.-

Methods to study stress resistance Biochemical Approach control vs. resistant plants control vs. induced conditions The Genetic Approach identify mutants with altered response suppressor mutations Comparative approach: complementation in yeast The Genomic Approach The Metabolomic Approach The Ionomic Approach Discovery vs. Hypothesis-Driven Science

Mendel Biotechnology was founded in 1997 to develop the idea that controlling gene expression would improve plant growth and development. Mendel focuses on a large class of genes called transcription factors because they control the degree to which each gene in a cell is activated. The approximately 27,000 genes in the Arabidopsis genome are controlled by approximately 1,800 different transcription factors. By systematically analyzing the function of all Arabidopsis transcription factors, Mendel scientists have discovered that single transcription factors can control complex traits such as freezing or drought, yield, morphology, disease resistance, nitrogen use efficiency and many other complex traits. We believe that no other company or academic institution in the world has a comparable understanding of the function of transcription factors and that Mendel is the leading company in the world in this area. The company has filed a large number of patents. We believe that the inventions described in our patent filings have placed the company in a strong position to participate in future developments in plant biotechnology, plant breeding, horticulture, and forestry. We are also using knowledge of plant gene expression to discover new ways of using chemicals to control plant growth and development.

Other Products HAYWARD, California--October 2005 Mendel Biotechnology Awarded SBIR Phase II Grant for Continued Research on Wide Spectrum Disease Resistance HAYWARD, California--October 2005 Mendel's role in Monsanto trait development program highlighted in September 2005 issue of Business 2.0 HAYWARD, California--September 2005 Mendel Biotechnology and SweTree Technologies will jointly exploit the value of certain genes in forestry HAYWARD, California--October 29, 2004 Both companies announced today that they will jointly exploit the value of certain transcription factor (TF) genes within the $750 billion forestry field, among them the Hercules gene. The collaboration includes a research effort to validate certain Mendel lead TF genes for the forestry field. Mendel has demonstrated that the Hercules gene and other TF genes improve growth rate in commercial plants. Mendel Biotechnology Awarded SBIR Phase II Grant for Continued Research to Increase Production of Natural Rubber HAYWARD, California--(PRNewswire)--October 7, 2003 The Economist writes about Mendel's offer to donate drought-protection technology to the Rockefeller Foundation. -- March 27, 2003 Other Products Disease resistance Abiotic stress tolerance Yield Flowering time Pigmentation Morphology Mineral nutrient use Metabolite composition Biomass composition

The complexity of stress adaptation: major targets for engineered stress tolerance

Functional genomics approach

II. PLANT RESPONSES TO HORMONES Hormone = A compound produced by one part of an organism that is transported to other parts where it triggers a response in target cells and tissues. B. Plant hormones help coordinate growth, development, and responses to environmental stimuli 1) By affecting division, elongation, and differentiation of cells 2) Effects depend on site of action, stage of plant growth and hormone concentration 3) The hormone signal is amplified, perhaps by affecting gene expression, enzyme activity, or membrane properties 4) Reaction to hormones depends on hormonal balance 5) Five classes of plant hormones: (1) Auxin (such as IAA). (2) Cytokinins (such as zeatin) (3) Gibberellins (such as GA3) (4) Abscisic acid (5) Ethylene

hormones are chemical signals that are produced in one part of the body, transported to other parts, bind to specific receptors, and trigger responses in targets cells and tissues. Only minute quantities of hormones are necessary to induce substantial change in an organism. Often the response of a plant is governed by the interaction of two or more hormones.

Plant hormones are produced at low concentration Signal transduction pathways amplify the hormonal signal many fold and connect it to a cell’s specific responses. These include altering the expression of genes, by affecting the activity of existing enzymes, or changing the properties of membranes. Response to a hormone usually depends not so much on its absolute concentration as on its relative concentration compared to other hormones

Stress physiology... ~50:50 %, thus not just a barrier Biological membranes are the primary target of many environmental stresses. Membranes are made of phospholipids and proteins. ~50:50 %, thus not just a barrier phospholipid hydrophobic interior phospholipid hydrophilic exterior