Preface   Physiology is one of the fundamental subjects of life science and knowledge of its practical aspects, particularly the biochemical aspect(s) is absolutely essential for the post graduate students as well as for researcher. We have always felt the necessity for a manual on orientation towards biochemical aspect of practical plant physiology. The present book is written in simple language covering the various aspects of basic and a clear-cut practical concept has been offered in the manual in respect of all quantitative experiments. Biochemical methods are used in all branches of biological science including agriculture. Biochemical aspect is an integral part of plant physiology and this aspect is used to explain nearly all the phenomenon of physiological aspect of plant and/or crop. Technology and Methods for Biochemical Aspects of Plant Physiology is mainly intended for Post Graduate students and Researchers of Universities and of different Research Institutes. As it covers a broad range of subjects on the basic as well as the practical aspects of biochemical part of Plant Physiology, it is likely that it will be also useful for any student attending different theoretical or practical Plant Physiology as well as Biochemistry courses. The book builds on pre-existing knowledge obtained at under graduate classes for different aspects of Biochemistry and Plant Physiology. It assumes a solid background and experience in handling of chemical as well as biochemicals. This book discuss the theoretical principles and the practical. This book has the description of different biochemical estimations, and it contains detailed experimental protocol(s) to perform experiments. It rather contains a collection and description of principles that will help the students perform successful experiments on biochemical aspect of plant physiology on their own during their future carrier. The experience of the author gathered during several years of service at the Indian Institute of Pulses Research, Kanpur resulted in a material that enables students to efficiently use practical on Biochemical Aspects of Plant Physiology later during their training. Moreover, the present book also provides a solid background on basic aspects biochemical estimations, a prerequisite for successful experimental design.

Plant physiology is a sub-discipline of botany concerned with the functioning, or physiology of plants. Closely related fields include plant morphology (structure of plants), plant ecology (interactions with the environment), phytochemistry (biochemistry of plants), cell biology, genetic, biophysics and molecular biology. Fundamental processes such as photosynthesis, respiration, plant nutrition, plant hormone functions, tropism, nastic movements, photoperiodism, photo-morphogenesis, circadian rhythms, environmental stress physiology, seed germination, dormancy, stomata function and transpiration, both parts of plant water relations, are studied by plant physiologists. The field of plant physiology includes the study of all the internal activities of plants those chemical and physical processes associated with life as they occur in plants. This includes study at many levels of scale of size and time. At the smallest scale are molecular interactions of photosynthesis and internal diffusion of water, minerals, and nutrients. Major sub disciplines of plant physiology include phytochemistry (the study of the biochemistry of plants) and phytopathology (the study of disease in plants). The scope of plant physiology as a discipline may be divided into several major areas of research.

Tissue or cell samples from a living organism, different cell cultures grown in a laboratory incubator under controlled conditions, homogenates or extracts of cells and tissues, solutions of isolated and purified components (e.g. proteins, nucleic acids) can all be referred to as biological samples. As the medium of life is water, the majority of biological samples can be defined as aqueous solutions with one or more components, colloidal systems, or water-based suspensions (e.g. bacterial cells dispersed in a liquid medium). Consequently, most biochemical experiments also take place in aqueous environments. Different solids (e.g. chemical substances obtained from different companies, synthetic oligonucleotides or peptides) are also often necessary for biochemical research. In most cases, solids are dissolved in water (or sometimes in other solvents) prior to the experiments. Therefore, the methods of preparing solutions and measuring accurately the weight of the required solids will also be discussed below. Sometimes we use gases in the laboratory. These can be stored in gas cylinders (e.g. O2), in Dewar flasks in liquid state (e.g. liquid nitrogen), or dissolved in water (e.g. HCl or NH3). By working with gases it is very important to follow all safety instructions to avoid fire, explosions, frostbite or (in case of inhalation) asphyxia or poisoning.

Units/measures are used to specify quantities. Every unit has a well-defined standard basic value, which is used as a reference in measurements. The units used in scientific practice constitute a scientifically established system, SI (Système International). Although only SI units are official , numerous non-SI units are still widely used (calorie, Ångström, Celsius etc.). We distinguish two types of quantities with two corresponding types of SI units. The base quantities cannot be derived from other quantities. Many of these are used in biochemistry (names of units and their symbols are given in brackets) to quantify the mass (gram, g), the length (meter, m), the time (second, s), the temperature (kelvin, K) and the charge (coulomb, C). Much larger is the group of derived quantities. Of these, the various units of concentration (to characterise the abundance of materials in solutions and other mixtures), volume and energy are the ones most frequently used in biochemistry. It is important to note that the terms mass and weight are often used interchangeably as alternatives. Technically, however, they have different meanings. The mass is the total quantity of matter in an object, which comes from the mass of all of its protons and neutrons, although not simply additively. Weight is a measure of the gravitational force exerted on an object. As the mass of protons and neutrons is the unit mass, their total mass in an object (e.g. the mass of a molecule, the molar mass) is a unitless number (e.g. the molar mass of water is 18). In other words, the molar mass is a relative number that would reflect the number of protons and neutrons in an atom or molecule, if the masses of protons and neutrons were additive. We make mass measurable by expressing it in grams because this way we can handle it as weight and measure it with a balance, the device developed for this purpose. 

As homogeneity is a basic criterion, it is important to know the solubility of solutes, which is a temperature dependent property. Besides temperature, solubility is influenced by the acidity (pH) of the solvent due to the acid-base character of the solute. Bases tend to dissolve better in acidic solutions, whereas acids do in basic solutions. Therefore, one often needs to change the pH of solutions accordingly. As the most widely used concentration units (molarity, w/v% and v/v%) refer the amount of solute in a given volume of the solution, it is the volume of the complete solution that must be set to the desired (calculated) value thus, one must consider not only the solvent but the solute(s) as well. Another reason why one must add the solute(s) to an amount of solvent which is less than the final volume might be that one will need to set the pH subsequently in order to enhance solubility and/or to reach a desired pH value. The volume of acid or base solutions used for pH setting is usually not known in advance and is not negligible. If the solute was initially dissolved in the final desired volume, one would exceed this volume during pH setting (even if the volume of the solute is negligible) and, thus, the solution would not have the desired (calculated) concentration but a smaller (and mostly unknown) one. Thus, according to the correct procedure, one must take into consideration the fact that the final volume will contain the volume of the buffering component. value particularly in the case of dissolving proteins, which have a tendency to stick to the bottom of the dish, thereby making dissolution substantially slower.

A buffer is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. Its pH changes very little when a small amount of strong acid or base is added to it and thus it is used to prevent changes in the pH of a solution. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications. Many life forms thrive only in a relatively small pH range so they utilize a buffer solution to maintain a constant pH. One example of a buffer solution found in nature is blood.

Carbon assimilation or carbon fixation is the conversion of inorganic carbon (carbon dioxide) to organic compounds by living organisms. The most prominent example is photosynthesis, although chemosynthesis is another form of carbon fixation that can take place in the absence of sunlight. Organisms that grow by fixing carbon are called autotrophs. Autotrophs include photoautotrophs, which synthesize organic compounds using the energy of sunlight, and lithoautotrophs, which synthesize organic compounds using the energy of inorganic oxidation. Heterotrophs are organisms that grow using the carbon fixed by autotrophs. The organic compounds are used by heterotrophs to produce energy and to build body structures. “Fixed carbon”, “reduced carbon”, and “organic carbon” are equivalent terms for various organic compounds.

A carbohydrate is a large biological molecules, or macromolecules, consisting of carbon (C), hydrogen (H), and oxygen (O) atoms, usually with a hydrogen : oxygen atom ratio of 2:1 (as in water); in other words, with the empirical formula Cm(H2O)n (where m could be different from n). Some exceptions exist; for example, deoxyribose, a sugar component of DNA, has the empirical formula C5H10O4. Carbohydrates are technically hydrates of carbon; structurally it is more accurate to view them as polyhydroxy aldehydes and ketones. The term is most common in biochemistry, where it is a synonym of saccharide. The carbohydrates (saccharides) are divided into four chemical groups: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. In general, the monosaccharides and disaccharides, which are smaller (lower molecular weight) carbohydrates, are commonly referred to as sugars. While the scientific nomenclature of carbohydrates is complex, the names of the monosaccharides and disaccharides very often end in the suffix -ose. For example, grape sugar is the monosaccharide glucose, cane sugar is the disaccharide sucrose, and milk sugar is the disaccharide lactose.

Nitrogen assimilation is the formation of organic nitrogen compounds like amino acids from inorganic nitrogen compounds present in the environment. Organisms like plants, fungi and certain bacteria that cannot fix nitrogen gas (N2) depend on the ability to assimilate nitrate or ammonia for their needs. Other organisms, like animals, depend entirely on organic nitrogen from their food. Plants absorb nitrogen from the soil in the form of nitrate (NO3 -) and ammonia (NH4). In aerobic soils where nitrification can occur, nitrate is usually the predominant form of available nitrogen that is absorbed. However this need not always be the case as ammonia can predominate in grasslands and in flooded, anaerobic soils like rice paddies. Plant roots themselves can affect the abundance of various forms of nitrogen by changing the pH and secreting organic compounds or oxygen. This influences microbial activities like the interconversion of various nitrogen species, the release of ammonia from organic matter in the soil and the fixation of nitrogen by non-nodule-forming bacteria.

Proteins are large biological molecules, or macromolecules, consisting of one or more chains of amino acid residues. Proteins perform a vast array of functions within living organisms, including catalysing metabolic reactions, replicating DNA, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in folding of the protein into a specific three-dimensional structure that determines its activity. A polypeptide is a single linear polymer chain derived from the condensation of amino acids. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids; however, in certain organisms the genetic code can include selenocysteine and, in certain archaea-pyrrolysine. Shortly after or even during synthesis, the residues in a protein are often chemically modified by post translational modification, which alters the physical and chemical properties, folding, stability, activity, and ultimately, the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can also work together to achieve a particular function, and they often associate to form stable protein complexes. Like other biological macromolecules such as pollysaccharides and nucleic acids, proteins are essential parts of organisms and participate in virtually every process within cells. Many proteins are enzymes that catalyse biochemical reactions and are vital to metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signalling, immune responses, cell adhesion, and the cell cycle. Proteins are also necessary in animals’ diets, since animals cannot synthesize all the amino acids they need and must obtain essential amino acids from food. Through the process of digestion, animals break down ingested protein into free amino acids that are then used in metabolism.

Amino acids are biologically important organic compounds composed of amine (-NH2) and carboxylic acid (-COOH) functional groups, along with a side-chain specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, though other elements are found in the side-chains of certain amino acids. About 500 amino acids are known and can be classified in many ways. They can be classified according to the core structural functional groups’ locations as alpha- (?-), beta- (β), gamma- (?), or delta- (δ-) amino acids; other categories relate to polarity, pH level, and side-chain group type (aliphatic, acrylic, aromatic, containing hydroxyl or sulphur, etc.). In the form of proteins, amino acids comprise the second-largest component (water is the largest) of human muscles, cells and other tissues. Outside proteins, amino acids perform critical roles in processes such as neurotransmitter transport and biosynthesis. Amino acids having both the amine and the carboxylic acid groups attached to the first (alpha-) carbon atom have particular importance in biochemistry. They are known as 2-, alpha-, or ?-amino acids (generic formula H2NCHRCOOH in most cases where R is an organic substituent known as a “side-chain”); often the term “amino acid” is used to refer specifically to these. They include the 22 proteinogenic (“protein-building”) amino acids, which combine into peptide chains (“polypeptides”) to form the building-blocks of a vast array of proteins. These are all L-stereoisomers (“left-handed” isomers), although a few D-amino acids (“right-handed”) occur in bacterial envelopes and some antibiotics. Twenty of the proteinogenic amino acids are encoded directly by triplet codons in the genetic code and are known as “standard” amino acids. The other two (“non-standard” or “non-canonical”) are pyrrolysine (found in methanogenic organisms and other eukaryotes) and selenocysteine (present in many noneukaryotes as well as most eukaryotes). For example, 25 human proteins include selenocysteine (Sec) in their primary structure, and the structurally characterized enzymes (selenoenzymes) employ Sec as the catalytic moiety in their active sites. Pyrrolysine and selenocysteine are encoded via variant codons; for example, selenocysteine is encoded by stop codon and SECIS element. Codon–tRNA combinations not found in nature can also be used to “expand” the genetic code and create novel proteins known as alloproteins incorporating non-proteinogenic amino acids.

Lipids are a group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, phospholipids, and others. The main biological functions of lipids include storing energy, signalling, and acting as structural components of cell membrane. Lipids have applications in the cosmetic and food industries as well as in nanotechnology. Lipids may be broadly defined as hydrophobic or amphiphilic small molecules; the amphiphilic nature of some lipids allows them to form structures such as vesicles, liposomes, or membranes in an aqueous environment. Biological lipids originate entirely or in part from two distinct types of biochemical subunits or “building-blocks”: ketoacyl and isoperen groups. Using this approach, lipids may be divided into eight categories:fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids and polyketides (derived from condensation of ketoacyl subunits); and sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Although the term lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides and phospholipids), as well as other sterol containing metabolites such as chloestrol. Although humans and other mammals use various biosynthetic pathways to both break down and synthesize lipids, some essential lipids cannot be made this way and must be obtained from the diet.

Plant nutrition is the study of the chemical elements and compounds that are necessary for plant growth, and also of their external supply and internal metabolism. The criteria for an element to be essential for plant growth: In its absence the plant is unable to complete a normal life cycle; or That the element is part of some essential plant constituent or metabolite.

Biological pigments, also known simply as pigments or biochromes are substances produced by living organisms that have a colour resulting from selective colour absorption. Biological pigments include plant pigments and flower pigments. Many biological structures, such as skin, eyes, fur and hair contain pigments such as melanin in specialized cells called chromatophores. Pigment colour differs from structural colour in that it is the same for all viewing angles, whereas structural colour is the result of selective reflection or iridescence, usually because of multilayer structures. For example, butterfly wings typically contain structural colour, although many butterflies have cells that contain pigment as well.

In organic chemistry, phenols, sometimes called phenolics, are a class of chemical compounds consisting of a hydroxyl group (—OH) bonded directly to an aromatic hydrocarbon group. The simplest of the class is phenol, which is also called carbolic acid C6H5OH. Phenolic compounds are classified as simple phenols or polyphenols based on the number of phenol units in the molecule.

Plant secondary metabolism produces products that aid in the growth and development of plants but are not required for the plant to survive. Secondary metabolism facilitates the primary metabolism in plants. This primary metabolism consists of chemical reactions that allow the plant to live. In order for the plants to stay healthy, secondary metabolism plays a pinnacle role in keeping all the of plants’systems working properly. A common role of secondary metabolites in plants is defense mechanisms. They are used to fight off herbivores, pests, and pathogens. Although researchers know that this trait is common in many plants it is still difficult to determine the precise role each secondary metabolite. Secondary metabolites are used in anti-feeding activity, toxicity or acting as precursors to physical defense systems.

Plant hormones (also known as phytohormones) are chemicals that regulate plant growth, which are termed ‘plant growth substances’. Plant hormones are signal molecules produced within the plant, and occur in extremely low concentrations. Hormones regulate cellular processes in targeted cells locally and, when moved to other locations, in other locations of the plant. Hormones also determine the formation of flower, stems, leaves, the shedding of leaves, and the development and ripening of fruit. Plants, unlike animals, lack glands that produce and secrete hormones. Instead, each cell is capable of producing hormones. Plant hormones shape the plant, affecting seed growth, time of flowering, the sex of flowers, senescence of leaves, and fruits. They affect which tissues grow upward and which grow downward, leaf formation and stem growth, fruit development and ripening, plant longevity, and even plant death. Hormones are vital to plant growth, and, lacking them, plants would be mostly a mass of undifferentiated cells. So they are also known as growth factors or growth hormones.. Phytohormones are found not only in higher plants, but in algae too, showing similar functions, and in microorganisms, like fungi and bacteria, but, in this case, they play no hormonal or other immediate physiological role in the producing organism and can, thus, be regarded as secondary metabolites.

Nucleic acids are polymeric macromolecules, or large biological molecules, essential for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. Each nucleotide has three components: a 5-carbon sugar, a phosphate group, and a nitrogenous base. If the sugar is deoxiribose, the polymer is DNA. If the sugar is ribose, the polymer is RNA. Together with proteins, nucleic acids are the most important biological macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information—in other words, information is conveyed through the nucleic acid sequence, or the order of nucleotides within a DNA or RNA molecule. Strings of nucleotides strung together in a specific sequence are the mechanism for storing and transmitting hereditary, or genetic, information via protein synthesis. The term nucleic acid is the overall name for DNA and RNA, members of a family of biopolymers, and is synonymous with polynucleotide. Nucleic acids were named for their initial discovery within the nucleus, and for the presence of phosphate groups (related to phosphoric acid). Although first discovered within the nucleus of eukaryotic cells, nucleic acids are now known to be found in all life forms as well as some nonliving entities, including within bacteria, archaea, mitochondria, chloroplasts, viruses and viroids. All living cells contain both DNA and RNA (except some cells such as mature red blood cells), while viruses contain either DNA or RNA, but usually not both. The basic component of biological nucleic acids is the nucleotide, each of which contains a pentose sugar (ribose or deoxyribose), a phosphate group, and a nucleobase. Nucleic acids are also generated within the laboratory, through the use of enzymes (DNA and RNA polymerases) and by solid-phase chemical synthesis. The chemical methods also enable the generation of altered nucleic acids that are not found in nature, for example peptide nucleic acids.

A vitamin is an organic compound required by an organism as a vital nutrient in limited amounts. An organic chemical compound (or related set of compounds) is called a vitamin when it cannot be synthesised in sufficient quantities by an organism, and must be obtained from the diet. Thus, the term is conditional both on the circumstances and on the particular organism. For example, ascorbic acid (vitamin C) is a vitamin for humans, but not for most other animals. Supplementation is important for the treatment of certain health problems but there is little evidence of benefit when used by those who are otherwise healthy. By convention, the term vitamin includes neither other essential nutrients, such as dietary minerals, essential fatty acids, or essential amino acids (which are needed in larger amounts than vitamins) nor the large number of other nutrients that promote health but are otherwise required less often. Thirteen vitamins are universally recognized at present. Vitamins are classified by their biological and chemical activity, not their structure. Thus, each “vitamin” refers to a number of vitamer compounds that all show the biological activity associated with a particular vitamin. Such a set of chemicals is grouped under an alphabetized vitamin “generic descriptor” title, such as “vitamin A”, which includes the compounds retinal, retinol, and four known carotenoids. Vitamers by definition are convertible to the active form of the vitamin in the body, and are sometimes inter-convertible to one another, as well. Vitamins have diverse biochemical functions. Some, such as vitamin D, have hormone-like functions as regulators of mineral metabolism, or regulators of cell and tissue growth and differentiation (such as some forms of vitamin A). Others function as antioxidants (e.g., vitamin E and sometimes vitamin C).