461N. Tuteja and S. Singh Gill (eds.), Plant Acclimation to Environmental Stress, DOI 10.1007/978-1-4614-5001-6_17, Springer Science+Business Media New York 2013
Plants are important natural resources source which can convert the solar energy into various usable forms including energy substance glucose and storage carbohydrate like starch. However, time to time, the potential of plants is challenged by many environmental uctuations including various biotic and abiotic stresses. Excess and de cit of any physical or biological factor can cause stress to the plant. Recently, the world is witnessing climatic changes such as increase in temperature, irregular rains, drought, excessive salt, ooding, excessive chilling, cyclones, tornedos, and various other natural calamities.
Stress causes changes in plant cellular functioning; as it prepares the plant for the incoming dangers. All the inactive protector genes become active due to sudden shift in the biochemistry and molecular biology of plant cell. The key organ in plants that acts as a rst line of defense is cell wall. Which is useful to protect plant from pest or pathogen invasion and chilling or mechanical stress. However, it is unable to secure plants from stresses like water de cit, salt stress etc.
D. Bhardwaj International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg , New Delhi , 110 067 , India
Department of Botany , University of Delhi , New Delhi , India
S. Lakhanpaul Department of Botany , University of Delhi , New Delhi , India
N. Tuteja (*) Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg , New Delhi , India e-mail: email@example.com
Chapter 17 Can G-Proteins be the Key Proteins for Overcoming Environmental Stresses and Increasing Crop Yield in Plants?
Deepak Bhardwaj , Suman Lakhanpaul, and Narendra Tuteja
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Recently, G-proteins and related machinery has been noted for its role in regulating stress related pathways, including ROS production, stomatal regulation and process related to plant water relations. We hereby present the role of G-proteins in all aspects of plant life and its metabolism (AM Jones 1994 ).
2 What is Stress? Perception and Signal Transduction Pathway
Any object that is affected by stress whether living or non-living undergoes de nite change in response to stress which is known as strain. In case of plants it is hard to measure the propensity of stress. However, during stressed conditions plants show many molecular and cellular changes.
Water is a primary requirement of plant, and its excess as well as de cit affect the growth of plants. Water is absorbed by roots, and root hairs serve as channels to increase the surface area of absorption. The whole physiology of stress can only be understood if one knows the parameters that decide the water status of plant. These parameters are water potential and relative water content.
Presence of cell wall in case of plants protects them from outer dangers including pathogens and adverse conditions. Plant cell membrane is a semi-permeable, quasi- uid structure which can allow only selective substances to pass through it; they serve as a second wall having many gates in the form of receptors and ion channels. Receptors being sensitive in nature are vulnerable to several elicitors that direct plant cell to respond against various stimuli. The cellular responses are initiated primarily by interaction of the extracellular material with a plasma membrane pro-tein. This extracellular molecule is called a ligand (or an elicitor) and the plasma membrane protein, which binds and interacts with this molecule, is called a recep-tor. Interestingly, various stress factors, both abiotic as well as biotic, act as elicitors for the plant cell. Plant growth and development are mediated by complex array of signaling pathways co-ordinated by exogenous factors, which regulate all phases of growth including cell division, differentiation, and cell death. During evolution all living organisms including plants have been subjected to continuous variation of external environmental stimuli such as heat, cold, light, darkness, and other rhyth-mic parameters. In response to these external stimuli cells start many reactions to regulate their physiological processes. The cells of multicellular organisms also need to communicate with each other in order to co-ordinate their growth and dif-ferentiation. All fundamental processes of biology including growth and develop-ment rely on the proper response to environmental signals (Bowler and Chua 1994 ). The molecular mechanism by which these signals are perceived by cells, transduced toward the proper targets, and integrated into a biological response is known as signal transduction. Basically, two functional principles are involved in the extracel-lular signal (primary stimulus) as the rst messenger itself penetrates the cell (prob-ably through speci c receptors) and nds its way to the nucleus. Alternatively, the signal remains outside the cell and is converted at plasma membrane into intracellular
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signals called second messenger (Hucho and Buchner 1997 ). Subsequent, sites are in the cytoplasm, at organelle membranes such as nuclear envelope and inside the nucleus.
Cell is like a universe where proteins like humans interact with each other, and one protein can interact with several and so on. These connections and interactions create a network or web of many signaling cascades which nally lead to the dif-ferential regulation of genes. Receptors are like sensors that sense the external envi-ronment and send the information to the cell machinery through various effectors, carriers, modulators, transcription factors, secondary messengers, enzymes etc. No information can be transcribed into response until receptor sends the signals to nucleus which is harboring plethora of dormant genes. Many plant receptors have been reported till date.
Stress is rst recognized by the receptors present on the plasma membranes. Many membrane receptors are characterized till date which includes serine threo-nine kinase (Afzal et al . 2008 ) , receptor-like protein kinase (Walker 1994 ) , cal-cineurin B-like sensors (Cheong et al . 2007 ) , G-protein-coupled receptor (Jones and Assmann 2004 ) , etc. After recognition, there is an induction of the relay of various proteins along with the generation of second messengers including cal-cium, calmodulin, calcium-induced protein kinases, calcium-dependent protein kinases, G-proteins, MAP kinase, (ROS), and inositol phosphates. Various pro-teins in a given cell may or may not work in isolation; they interact with several other proteins to bring about certain changes that could be the need of time. This initiates the activity of various kinases and phosphatases which ultimately targets various genes and trans-elements. The up-regulation of certain dormant genes is required for the survival of cell under stressed conditions. This gives plant the potential to adapt or acclimatize to the particular environment. There are many hormones related to stress that add to this effect and activate many stress-induced genes. Their level generally increases during stressed conditions and they further start different signaling pathways to resist stressed conditions.
There is a different group of molecules that initiates and catalyses the modi cation of certain signaling proteins. They help in the modi cation or assembly of signaling components. Myristoylation, glycosylation, methylation and ubiquitination are some common modi cations that give stress-related proteins some special features so that they can perform functions under alternate situations. Stress response could be early or late, and, on the basis of this, various stress-responsive genes can be broadly categorized as early and late induced genes. Early genes are induced within minutes and these genes belong to transcription factors, whereas other genes like RD (responsive to dehydration) (Kariola et al . 2006 ) and KIN (cold induced)/COR (cold responsive) (Thomashow 1998 ) , which encode and modulate the proteins needed for synthesis of LEA-like proteins (late embryogenesis abundant) ( Hundertmark 2008 ) , antioxidants, membrane-stabilizing proteins. However syn-thesis of osmolytes take hours to get induced.
There are many signature elements on the promoter of genes that also tell the importance of particular gene in the alleviation of various stresses such as. DRE (dehydration-responsive elements) (Sun et al . 2008 ) or CRT (C-repeats) and some
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of them which contain ABRE (ABA-responsive element) ( Gmez-Porras et al . 2007 ) . Stress-induced transcription factors bind to this element and their overex-pression brings about the tolerance against various stresses. Same way, there are many nuclear genes whose overexpression can give genetic potential to the plant against any kind of environmental challenges.
Description of all the genes of all the genes, transcription factors, chaperones and related proteins are beyond the scope of this chapter and we will only focus on the G-proteins and their role in various plant processes including stress.
3 What are G-proteins and Their Role in Plant Metabolism, Growth, and Development?
3.1 GPCR and G-proteins
Plant membranes like animal membranes are studded by G-protein-coupled receptor (GPCR), RGS and heterotrimeric G-proteins (G a , G b /G g subunits) that are often link to the GPCR, constituting one of the most important components of cell signal-ing cascade. Signals are perceived by GPCR or RGS and later on transduced to inner part of the cell through G-proteins. This constitutes GPCR/G-protein-induced signaling cascade. Alfred G. Gilman and Martin Rodbell were awarded Nobel prize in 1994 for their contribution in the eld of cell signal transduction; they proved the role of G-proteins as signaling molecules. Martin Rodbell and his collaborators linked the transducer with hormones, whereas Alfred G. Gilman and his co-worker characterized and puri ed the G-protein. Hundreds of chemicals and physical sig-nals that can be stress-causing elicitors constantly bombard the surface of all cells. Some of these do not enter the cell, but, instead, bind to receptors at the cell surface and initiate a ow of information that moves to the cell interior. The receptors for many hormones (such as catecholamine, gonadotropin, parathyroid hormone, glu-cagon and ABA), odorants and light are heptahelical structures. Stimulation of these receptors causes activation of special proteins that are linked to the G-proteins. G-protein performs various functions including regulating few types of enzymes and ion channel. The target enzymes or ion channels are called effectors and they are generally proteinaceous in nature; G-protein after activation becomes effector itself and it interacts with other proteins to bring about changes in their activity causing alterations in ionic composition or in second messenger level that ultimately leads to the cellular response (Neer 1995 ) .
GPCR is an integral membrane protein receptor that contains seven-trans-membrane a -helical regions and this portion of receptor binds to a wide range of ligands like hormones. Interestingly, binding of ligands can bring about confor-mational changes in the structure of GPCR activating G-proteins. Binding of ligands is a switching on of various steps that happen in tandem. This includes the
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exchange of GDP/GTP associated with the G a subunit and disassociation of alpha subunit from beta gamma dimer. The separated subunits individually act as effec-tors and interact with other intracellular proteins. This whole process can be switched off by simply replacing GTP attached to alpha subunit by GDP. This creates the earlier situation when the heterotrimer of G-proteins was attached to GPCR as a dormant unit. Nevertheless, GPCR can also work independent of G-proteins (AM Jones 2002 ) .
3.2 Historical Background of G-proteins
Historically, it is work on adenylyl cyclase since 1950 that nally resulted in the discovery of G-protein. Now, it is well known that G-proteins are involved in broad range of cellular regulatory activities. Nobel Prizes has been awarded in this eld to Sutherland and Rall ( 1958 ) , who discovered the cAMP and adenylyl cyclase as a second messenger; Fischer and Krebs (1992), whose extensive studies of reversible protein phosphorylation began with cAMP-dependent protein kinase; and Gilman and Rodbell (1994) for their work on heterotrimeric G-proteins (Vaugham 1998 ) . Here, a brief history of G-proteins has been described.
19571958 The G-proteins eld originated. Sutherland and Rail described the basic properties of the enzyme adenylyl cyclase (AC), which is involved in hormone (epinephrine)-regulated synthesis of second messenger (cAMP) (Rall 1957 ; Sutherland and Rall 1958 )
1969 Fat cell adenylyl cyclase was reported to be activated by multiple hormones: the hormone receptors were distinct from the enzyme. ATP could reverse the binding action of glucagon to the rat liver cell membrane receptor and thus dissociate the glucagon from the cell (Birnbaumer and Rodbell 1969 )
19711974 Role of GTP was rst reported by Martin Rodbell. GTP required for glucagon stimulated adenylyl cyclase in liver. GTP enhances disso-ciation of glucagon from its receptor (Rodbell et al. 1971 )
1975 Henery Bourne and co-workers isolated S49 mouse lymphoma cell line de cient in adenylyl cyclase activity, named cyc. These cells were later used as assay tool to isolate coupling factors (receptor, G-proteins, adenylyl cyclase) (Bourne et al. 1975 ) Pfeuffer and Helmreish ( 1975 ) separated a GTP-binding protein from the adenylyl cyclase complex
1976 Orly and Schramm ( 1976 ) directly demonstrated the independence of receptor and cyclase enzyme
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1977 E.M. Robs and A.G. Gilman in 1977 demonstrated the resolution of some components of adenylyl cyclase necessary for catalytic activity. They reported the 4G kDa GTP-binding protein, now, known as G
Rodbell discovered that G-proteins at the cell receptor could both inhibit and activate transduction, often at the same time
1978 Robert Lefkowitz in 1978 demonstrated the conformational change occurred to receptor after binding to ligands, and GTP regulated the association with macromolecule Cassel and Selingen in 1978 showed t...