Understanding plant abiotic stress responsesAbiotic stresses elicit complex cellular responses.Genetic engineering for increasing tolerance to abiotic stresses has come in due to the progress made in exploring and understanding plant abiotic stress responses at whole-plant( physiological, biochemical, cellular and molecular levels). Plant molecular biology is a fast-expanding research frontier of our times. This important branch of science has given several clues in understanding how plants respond under stressful regimes. A great deal of success has been achieved in unveiling gene/protein alterations associated with preparation of plants against the abiotic stresses. In parallel, major progress has been made in the characterization of stress-related promoters and transcription factors as well as stress signalling components.

Abiotic stresses-Environmental factorsFigure - Response of plants to lethal and sublethal level of stresses. The plant in an unfavourable environment could face the following two situations: lethal stress where the plant may ultimately die due to increased senescent activities (ii) Sublethal stress or lethal stress where certain adaptive changes may occur, leading to survival of the plant. These adaptations could be at the molecular level involving changes in gene expression, synthesis of stress proteins, etc. and at the biochemical level. The latter changes ultimately may bring about the physiological response and finally the whole plant response.

Environmental factors leading to plant diseasesThere are three types of responsess-1. Immediate response of invaded cells: 2.local response and gene inactivation: 3.Systemic response and gene inactivation1. Immediate response of invaded cells:(a) Generation of reactive oxygen species(b) Nitric oxide synthesis( c) Opening of ion channels(d)Protein phosphorylation/dephosphorylation (e)Cytoskeletal rearrangements(f) Hypersensitive cell death/response (HR)-necrosis(g) Gene induction

:2.local response and gene inactivation:Alterations in secondary metabolites/pathwaysCessation of cell cycleSynthesis of pathogen related proteinsAccumulation of benzoic and salicylic acid Production of ethylene and jasmonic acidFortification of cell-walls( lignin,PGIPs,HRGPs)3.Systemic response and gene inactivation: Glucanase,Chitinase,Peroxidases,Synthesis of other PR proteins

Responses to abiotic stresses-excess or deficit in physical/chemical environmentEnvironmental conditions- water logging, drought, high or low temperature, excessive soil salinity, inadequate mineral nutrients in the soil and too much or too little light, phytotoxic agents like ozone Environmental stresses depends on -Severity, duration,number of exposures ,combination of stresses Plant characteristics- organ or tissue, stage of development, genotype ResponseResistance/susceptibility

Result- survival & growth/death

Plant stresses greatly diminish crop yields

Average yield & Record yield of few major crops

CropRecord Yield (kg/hectare)Average Yield(kg/hectare)Average loss(kg/hectare) % Loss (kg/hectare)Corn19,3004,60012,70065.8wheat14,5001,88011,90082.1Soybean7,3901,6105,12069.3Oat10,6001,7207,96075.1Barley11,4002,0508,59075.4Potato94,10028,30050,90054.1Sugar beet121,00042,60061,30050.7Sorghum20,1002,83016,20080.6

---Resistance mechanisms allow to avoid or tolerate stress: Avoidance Prevent exposure to stress (e.g. phreatophytes-deep roots against drought avoidance-Honey mesquite,Mohave dessert star) Tolerance permits the plant to withstand the stress (e.g.Xerophytes- Saguaro Cactus)

Adaptations/Aclimation evolutionary improvements/adjustment of individual organisms by changing its homeostasis (e.g. Spinach (Osmotic adjustment) ,Black spruce (freezing tolerance)

Desert Ephemerals escape drought by germinating and completing their life-cycle while enough water is available (short lived-4-8 weeks)

Pictorials phreatophytes & xerophytesMohave desert starSaguaro

Pictorial-adaptaions/aclimationBlack spruceHoney mesquite (phreatophyte)Spinach

The word ephemeral means transitory or quickly fading (e.g.-Trillium grandifolium &Thalictrum thalicroides)

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 patternThese changes are integrated into a response by the whole plant that may modify growth ,development and even influence reproductive capabilitiesClues 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+) Cont..

ContTranscriptional activation of gene expression suggests that accumulation of gene products is also influenced by post transcriptional regulatory mechanisms that increase the amount of specific m RNAs,enhance translation,stabilizing proteins,alter protein activity or some combination of theseUsing molecular genetic techniques,scientists have started to dissect plant responses associated with exposure to specific stresses.

Model depicting various possible events involved in abiotic stressesstress 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

Stresses involving water deficitWater deficit can induced by many environmental conditionsTwo parameters that describe the water status of plants are -water potential and relative water contentWater potential is the potential energy of water per unit volume relative to pure water in reference conditions. Water potential quantifies the tendency of water to move from one area to another due to osmosis, gravity, mechanical pressure, or matrix effects such as surface tension. Water potential has proved especially useful in understanding water movement within plants, animals, and soil. Water potential is typically expressed in potential energy per unit volume and very often is represented by the Greek letter .

Components of water potential

Many different factors may affect the total water potential, and the sum of these potentials determines the overall water potential and the direction of water flow:/w = 0 + + p + s + v + m where:0 is the reference correction, is the solute potential, (no of particles dissolved in water)p is the pressure component, (physical forces exherting on water)s is the gravimetric component, v is the potential due to humidity, and m is the potential due to matrix effects (e.g., fluid cohesion and surface tension.) how solid surfaces like CW& colloids interact with waterAll of these factors are quantified as potential energies per unit volume, Movement of liquid water into or out of plant cell - /w = p + s High to low (PM,Tonoplast and membranes of organelles)Pressure chambers/thermocouple psychrometer measures /w

Pressure Potential

Pressure potential is based on mechanical pressure, and is an important component of the total water potential within plant cells. Pressure potential is increased as water enters a cell. As water passes through the cell wall and cell membrane, it increases the total amount of water present inside the cell, which exerts an outward pressure that is retained by the structural rigidity of the cell wall. By creating this pressure, the plant can maintain turgor, which allows the plant to keep its rigidity. Without turgor, plants lose structure and wilt.The pressure potential in a living plant cell is usually positive. In plasmolysed cells, pressure potential is almost zero. Negative pressure potentials occur when water is pulled through an open system such as a plant xylem vessel. Withstanding negative pressure potentials (frequently called tension) is an important adaptation of xylem vessels.Defined in units of pressure(megapascals) rather than energy

Solute potential

Pure water is usually defined as having a solute potential () of zero, and in this case, solute potential can never be positive. The relationship of solute concentration (in molarity) to solute potential is given by the van 't Hoff equation: = MiRTwhere M is the concentration in molarity of the solute, i is the van 't Hoff factor, the ratio of amount of particles in solution to amount of formula units dissolved, R is the ideal gas constant, and T is the absolute temperature.Cont..

Cont..when a solute is dissolved in water, water molecules are less likely to diffuse away via osmosis than when there is no solute. A solution will have a lower and hence more negative water potential than that of pure water. Furthermore, the more solute molecules present, the more negative the solute potential is.Solute potential has important implication for many living organisms. If a living cell with a smaller solute concentration is surrounded by a more concentrated solution, the cell will tend to lose water to the more negative water potential (w) of the surrounding environment. This is often the case for marine organisms living in sea water and halophytic plants growing in saline environments. In the case of a plant cell, the flow of water out of the cell may eventually cause the plasma membrane to pull away from the cell wall, leading to plasmolysis. It can be measured in plant cells using the Pressure bomb. Most plants, however, have the ability to increase solute inside the cell to drive the flow of water into the cell and maintain turgor.This effect can be used to power an osmotic power plant

Matrix potential (Matric potential)

When water is in contact with solid particles (e.g., clay or sand particles within soil), adhesive intermolecular forces between the water and the solid can be large and important. The forces between the water molecules and the solid particles in combination with attraction among water molecules promote surface tension and the formation of menisci within the solid matrix. Force is then required to break these menisci. The magnitude of matrix potential depends on the distances between solid particlesthe width of the menisci and the chemical composition of the solid matrix. In many cases, matrix potential can be quite large and comparable to the other components of water potential .It is worth noting that matrix potentials are very important for plant water relations. Strong (very negative) matrix potentials bind water to soil particles within very dry soils. Plants then create even more negative matrix potentials within tiny pores in the cell walls of their leaves to extract water from the soil and allow physiological activity to continue through dry periods. Germinating seeds have a very negative matrix potential. This causes water uptake in even somewhat dry soils and hydrates the dry seed.

The relative water content (RWC)The relative water content (RWC) stated by Slatyer in 1967 is a useful indicator of the state of water balance of a plant Relative water content (RWC) is the appropriate measure of plant water status in terms of the physiological consequence of cellular water deficit. While water potential as an estimate of plant water status is useful in dealing with water transport in the soil-plant-atmosphere continuum, it does not account for osmotic adjustment (OA). OA is a powerful mechanism of conserving cellular hydration under drought stress and RWC expresses the effect of OA in this respect. Hence RWC is an appropriate estimate of plant water status in terms of cellular hydration under the possible effect of both leaf water potential and OA.

Formula to calculate RWCRWC (%) = [(FW-DW) / (TW-DW)] x 100,Where,FW sample fresh weightTW sample turgid weightDW sample dry weight.Often reported in conjunction with Plant water potential (/w )RWC (less than 50%) below a critical value leads to tissue death

Osmotic stressUnder osmotic stress, accumulation of osmotically active compounds called osmolytes occur to lower the osmotic potential. These molecules are not highly charged, but are polar, highly soluble and have a larger hydration shell. Since s is a function of total no. of solutes in a given vol. of water, a remarkable array of organic compounds and inorganic ions contribute to the s In principle ,OA involve metabolic changes that alter rates of ion uptake, decrease assimilation of low mol.mass organic compounds, or enhance their synthesis

Important osmolytes that accumulate in plants during stressesCarbohydrate /Nitrogenous compound /Organic acidSucrose Proteins OxalateSorbitol Betaine MalateMannitol GlutamateGlycerol AspartateArabinitol GlycinePinitol CholineOther polyols Putrescine

Osmotic adjustment and its role in tolerance to drought and salinityOsmotic adjustment is a biochemical mechanism that helps plants acclimate to dry or saline soilCompatible solutes share specific biochemical attributesTransgenic plants can be used to test the acclimative functions of specific osmolytesGlycine betaine accumulation is regulated by the rates of its synthesis and transportIn some plant species ,salt stress inhibits sucrose synthesis and promotes accumulation of mannitolTaxonomically diverse plants accumulate pinitol in response to salt stress

Osmotic adjustment /Osmoregulation- a biochemical mechanismOsmotic adjustment /Osmoregulation is generally regarded as an important adaptation to drought or salinity.It helps to maintain turgor and cell volume, it is often thought to promote growth, yield, or survival, of plants in dry or saline soils.Plants extract water from the soil /w in roots surroundingsPlant roots must establish a /w gradient water flow towards the root from the soilWhen soil /w becomes too low- wilting (lose turgor)

Compatible solutes/osmolytes- specific biochemical attributesCompatible solutes are small group of chemically diverse organic compounds and their synthesis & accumulation is wide spread in plants but the distribution of these varies among plant speciesAmino acid Proline,( conc. maintained through a combo of synthesis & catabolism) accumulated in taxonomically diverse set of plants while -alanine betain in confined to Plumbaginaceae-leadwort

Role of osmolytes in plant protection against abiotic stressSome 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 PSIISorbitol,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

Glycine betain accumulation is regulated by the rates of its synthesis and transport

Ethanol amineATPADP Ethanol amine kinase Phosphoethanolamine/phosphatidylcholine s-adenosylmethionine phospho base N-methyltransferases-adenosylhomocysteinePhosphocholine-Pi Phosphocholine phosphatase Choline O2+2H+ +2Fdxox Choline monooxygenaseBetain aldehyde NAD+---NADH Betain aldehyde dehydrogenaseGlycine betain

Saline stress inhibits sucrose synthesis and promotes accumulation of mannitolFructose- 6-Po4 Phosphomannose isomerase

Mannose-6-Po4 Mannose -6 Po4 reductase NADPH----NADPMannitol-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-methyltransferaseS-adenosylhomocysteine Ononitol Ononitol epimerase Pinitol

Impact of water deficit and salinity on transport across plant membraneCarriers,pumps and channels operate to minimize the impact of perturbing ions on cell metabolismSynthesis and activity of aquaporin (proteinaceous transmembrane water channel in PM &tonoplast ) may be up-regulated in response to drought-e.g.RD28(MIP)in arabidopsis & -TIP(tonoplast intrinsic protein)undergo phosphorylation

Additional genes induced by water stressSome seed proteins may protect vegetative tissue from stressOsmotin,an alkaline protein ,accumulates during water deficitSome genes induced by water stress are responsive to ABASpecific cis-elements and trans- acting factors promote transcription in response to ABA and water deficit

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

Salt stress elicited Ca2+ signals are perceived by SOS3, whichactivates 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 byinhibiting SOS2 kinase activity or the activities of SOS2 targets. Double arrow indicates SOS3-independent and SOS2-dependent pathway.

ABA-dependent and ABA-independent signal transduction.

Stress-induced regulation of early and delayed response genes.

Environmental factors that increase concentration of ROSOzone- UV radiations +SO2,NO,NO2,--------------Stratasphoric/Troposphoric-Drought, Senesence,Herbicides,(paraquat dichloride) Wounding, Intense light, --OXIDATIVE STRESSPathogens,Heat&Cold,Heavy metals,Root nodules-----------------------------------------

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

Lea (Late embryogenesis abundant) GenesGenes induced in seeds during maturation and desiccation &also in vegetative tissues of plants in stresses(water deficit)Hydrophillic, rich in alanine & glycine, lacking cysteine & tryptophaneImportant due to their abundance & expression patterns while in-vivo activities unknownExamples-over expression in rice & yeast enhance resistance

Groups of Lea Genes/ProteinsGroup 1(D-19 family)-EM(early methionine labeled protein of Wheat)Group 2(D-11 family)- DHN1(maize) & D-11 (cotton)Group 3(D-7 family)- HVA1(ABA induced in Barley) &D-7 in cottonGroup 4(D-95 family)- D-95 in soybeanGroup 5(D-113 family)- LE25 (tomato) &D-113 (cotton

Freezing stressSome plants can acclimate to subfreezing temperatures- called cold acclimationA primary function of freeze- tolerance mechanism is membrane stabilizationRoles of the osmolytes and antifreeze proteins that accumulate in promoting freezing tolerance remain poorly understoodFreezing tolerance involves changes in gene expression

Flooding and oxygen deficitPlants vary in ability to tolerate floodingDuring short term acclimation to anoxic conditions,plants generate ATP through glycolysis and fermentationShifting from aerobic metabolism to glycolytic fermentation involves changes in gene expressionThe plant hormone ethylene promotes long-term acclimative responses,including formation of aerenchyma and stem elongation,in wetland and flood- tolerant speciesEthylene triggers epinasty in some flood sensitive speciesHow do plant sense oxygen deprivation

Impact of oxygen deprivation on respiratory metabolism

Normoxic (aerobic)-all ATP from oxidative phosphorylation-TCA-cycleHypoxic-Partial pressure of O2limits ATP-glycolusisAnoxic(anaerobic)-ATP by glycolysisLow protein synthesis,impaired cell division & elongation leads to cell death

Plant species categorized by sensitivity to Oxygen deprivationWetland plants- sweet flag, rice grass, coral tree, rice, wild rice, golden dock, barnyard grassFlood-tolerant plants- Arabidopsis, barnyard grass,oat,potato,wheat,cornFlood- sensitive plants- soy bean,tomato,peaStructural adaptationsAerenchyma (maize)-continuous,columnar intracellular spaces formed in root cortical tissuesLenticels (marshell)-opening in the periderm that allow gaseous exchangePneumatophores (mangrove)-shallow roots that grow with negative geotrophy out of aquatic environment

Oxidative stressTropospheric ozone is linked to oxidative stress in plantsOzone causes oxidative damage to biomoleculesChloroplasts are susceptible to ozone induced damageIncreased synthesis of antioxidants and antioxidant enzymes can improve tolerance to oxidative stressOxidative stress or ozone can interact with plant hormones such as SA and ethylene to produce plant responses

Heat StressHeat stress alters cellular functionsPlants can acclimate to heat stressHSPs are conserved among different organismsFive classes of HSPs are defined according to sizeExpression of many HSPs is controlled by a transcription factor that recognizes a conserved promotor sequences

Heat Shock Proteins (HSP)Response to heat stress decreases synthesis of normal proteins, but increases/accelerated transcription& translation of new set of proteins as HSP (5C above the optimal temp.)Plants can acquire thermo-tolerance if subjected to a nonlethal high temp. for a few hours before encountering heat shock conditions-involve the process of synthesis of HSP.Many of HSPs work as chaperons and are involved in refolding proteins denatured by heat HSPs are expressed through out development,& indeed some show sequence homology only while not induced during heat stress

Classes of HSPs

Protein ClassNameSize (kDa)LocationHSP100Hsp104(yeast)ClpB &ClpA ( E.coli)100-114cytoplasmHSP90Hsp90GRP9480-94Cytoplasm ERHSP70Dnak(E.coli)BiP & GRPSSA1-4(yeast)SSB1-2(yeast)SSC1(yeast)KAR2(yeast)69-71ERCytoplasmCytoplasmMitochondriaERHSP60Chaperonin 60groEL(E.coli)GroES(E.coli)605710Chloroplast & mitochondriasmHSP15-30Cytoplasm,chloroplast,ER,mitochondria

Heat Shock Transcription Factor(HSF)Expressed constitutely but must be activated by heat stress to recognize its DNA target i.e.HSEHSE is 5bp repeats in alternating origintations with the consensus nGAAn.An HSF regulated promoter may contain 5-7of these repeats close to TATA box.Many HSEs contain the DNA element 5-CTnGAAnnTTCnAG-3

(a) Generation of reactive oxygen species

Production of ROS (incompatible interactions)Typical ROS are super oxide (O2-) and hydrogen peroxide(H2O2)Production of superoxide from molecular oxygen involves plasma-membrane associated NADPH oxidase leads to production of H2O2 which is permeable to PM and highly toxicEventually H2O2 is converted into H2O by antioxidant enz., Plays a vital role in plant defense.

ROS-defense-mechanismH2O2 is directly toxic to pathogen & in presence of iron give rise to highly reactive hydroxyl radicle or may contribute to structural reinforcement of plant cell walls either by cross- linking various hydroxy-proline and proline- rich glycoproteins to the polysaccharide matrix or by increasing the rate of lignin polymerization by peroxidase enzyme activity and both would make the plant cell wall more resistant to microbial penetration and enzymatic degradationSignaling role of ROS: H2O2 induces benzoic acid 2- hydroxylase(BA 2-H) enzyme activity for SA biosynthesisH2O2 also induces genes for protein involved in certain cell protection mechanism(e.g.glutathione s- reductase. ROS production also alter the redox balance in responding cells (e.g.-specific plant transcription factors are redox regulated.