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What are the microscopic mechanisms of plant branching?

What are the microscopic mechanisms of plant branching?


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For a long time I've been idly interested in how the shape of a complex organism gets determined during development at a microscopic level. Recently I've realized that plants could be a good place to start trying to understand that, because the structure of even a quite complex-looking plant can sometimes be described by a few simple behaviors. Here are some examples.

  1. My radish plants have a single base where all foliage grows from. After it's first two seed leaves, it grows some number of large rough leaves which all descend to the same point. No other branching ever takes place.

  2. My pea plants have only one long stalk, which grows symmetrical pairs of leaves at regular intervals. Occasionally, the base of a leaf pair spawns a mini-stalk, never more than 3 or 4 leaf-pairs long, ending in a "claw" of three tendrils. Most of the time, one of the three tendrils will split again in three (always the one aligned with the "top" of the stem, as determined by leaf orientation) .

  3. My fennel plant has thick bases to its stems which each extend a few inches before branching starts. At that point, it branches at regular intervals in symmetric pairs of fronds, which in branch the same way. Those third-layer fronds branch at regular intervals but alternate sides, and there are always exactly four layers of branching no more and no less.

  4. My basil plants seem roughly able to grow branches in any advantageous spot, with no rigid rules or geometric behaviors like the other examples.

My background is in mathematics, and all my knowledge of biology is dated to high school. I've always been confused by how DNA is presented as a "list of proteins", when it is clearly so dynamical and not static: each cell type only produces some of those proteins, and those proteins can in turn determine what proteins it's children produce, I guess, and this can create a rich (non-linear) hierarchy of tissue differentiation out of a linear list.

For a really complex organism like a mammal I guess that our understanding of how DNA gets actualized is phenomenological (i.e. we can tell that gene X correlates with macroscopic trait Y, even if we have no idea what the signalling mechanisms are behind it and which cell types produce a relevant protein and when.)

But is this true also for plants, or do we have a deeper understanding of what proteins differentiate, for instance, the four stalk types of a fennel plant, and why each type can only produce the next one? I think simple animals like sponges and nematodes and cnidaria probably would be as helpful as (or more than) plants, but as a gardener I see these plant geometries every day and am fascinated by them.

Does anyone know of a good reference, book or online, that discusses these topics? I'm okay with something a little out of my technical depth - I'd prefer a difficult answer to an incomplete one.

EDIT: Leaf architecture is also interesting, and probably inseparable from branch architecture on some level, but I focused on stalks and branches probably because I don't have the terminology to meaningfully describe leaf architectures.


Types of Biology

The Greek words bios and logos are the origins of the term &ldquobiology&rdquo. Bios means life and logos means study. So, biology is the science which studies life and alive organisms. An organism means an alive entity having one cell such as bacteria, or several cells such as plants, fungi, and animals. Biology studies these living organisms&rsquo chemical processes, physical structure, physiological mechanisms, molecular interactions, evolution, and development. Biological science may seem very complex but some specific unifying concepts combine it into a single, consistent field. Biology specifies the cell as life&rsquos basic unit, genes as heredity&rsquos basic unit, and evolution as the generator that drives the species&rsquo creation and extinction.


Contents

Animal tissue classification Edit

There are four basic types of animal tissues: muscle tissue, nervous tissue, connective tissue, and epithelial tissue. [5] [9] All animal tissues are considered to be subtypes of these four principal tissue types (for example, blood is classified as connective tissue, since the blood cells are suspended in an extracellular matrix, the plasma). [9]

Plant tissue classification Edit

For plants, the study of their tissues falls under the field of plant anatomy, with the following four main types:

Histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. [5] [6] It is an important part of anatomical pathology and surgical pathology, as accurate diagnosis of cancer and other diseases often requires histopathological examination of tissue samples. [10] Trained physicians, frequently licensed pathologists, perform histopathological examination and provide diagnostic information based on their observations.

Occupations Edit

The field of histology that includes the preparation of tissues for microscopic examination is known as histotechnology. Job titles for the trained personnel who prepare histological specimens for examination are numerous and include histotechnicians, histotechnologists, [11] histology technicians and technologists, medical laboratory technicians, and biomedical scientists.

Most histological samples need preparation before microscopic observation these methods depend on the specimen and method of observation. [9]

Fixation Edit

Chemical fixatives are used to preserve and maintain the structure of tissues and cells fixation also hardens tissues which aids in cutting the thin sections of tissue needed for observation under the microscope. [5] [12] Fixatives generally preserve tissues (and cells) by irreversibly cross-linking proteins. [12] The most widely used fixative for light microscopy is 10% neutral buffered formalin, or NBF (4% formaldehyde in phosphate buffered saline). [13] [12] [9]

For electron microscopy, the most commonly used fixative is glutaraldehyde, usually as a 2.5% solution in phosphate buffered saline. [9] Other fixatives used for electron microscopy are osmium tetroxide or uranyl acetate. [9]

The main action of these aldehyde fixatives is to cross-link amino groups in proteins through the formation of methylene bridges (-CH2-), in the case of formaldehyde, or by C5H10 cross-links in the case of glutaraldehyde. This process, while preserving the structural integrity of the cells and tissue can damage the biological functionality of proteins, particularly enzymes.

Formalin fixation leads to degradation of mRNA, miRNA, and DNA as well as denaturation and modification of proteins in tissues. However, extraction and analysis of nucleic acids and proteins from formalin-fixed, paraffin-embedded tissues is possible using appropriate protocols. [14] [15]

Selection and trimming Edit

Selection is the choice of relevant tissue in cases where it is not necessary to put the entire original tissue mass through further processing. The remainder may remain fixated in case it needs to be examined at a later time.

Trimming is the cutting of tissue samples in order to expose the relevant surfaces for later sectioning. It also creates tissue samples of appropriate size to fit into cassettes. [16]

Embedding Edit

Tissues are embedded in a harder medium both as a support and to allow the cutting of thin tissue slices. [9] [5] In general, water must first be removed from tissues (dehydration) and replaced with a medium that either solidifies directly, or with an intermediary fluid (clearing) that is miscible with the embedding media. [12]

Paraffin wax Edit

For light microscopy, paraffin wax is the most frequently used embedding material. [12] [13] Paraffin is immiscible with water, the main constituent of biological tissue, so it must first be removed in a series of dehydration steps. [12] Samples are transferred through a series of progressively more concentrated ethanol baths, up to 100% ethanol to remove remaining traces of water. [9] [12] Dehydration is followed by a clearing agent (typically xylene [13] although other environmental safe substitutes are in use [13] ) which removes the alcohol and is miscible with the wax, finally melted paraffin wax is added to replace the xylene and infiltrate the tissue. [9] In most histology, or histopathology laboratories the dehydration, clearing, and wax infiltration are carried out in tissue processors which automate this process. [13] Once infiltrated in paraffin, tissues are oriented in molds which are filled with wax once positioned, the wax is cooled, solidifying the block and tissue. [13] [12]

Other materials Edit

Paraffin wax does not always provide a sufficiently hard matrix for cutting very thin sections (which are especially important for electron microscopy). [12] Paraffin wax may also be too soft in relation to the tissue, the heat of the melted wax may alter the tissue in undesirable ways, or the dehydrating or clearing chemicals may harm the tissue. [12] Alternatives to paraffin wax include, epoxy, acrylic, agar, gelatin, celloidin, and other types of waxes. [12] [17]

In electron microscopy epoxy resins are the most commonly employed embedding media, [9] but acrylic resins are also used, particularly where immunohistochemistry is required.

For tissues to be cut in a frozen state, tissues are placed in a water-based embedding medium. Pre-frozen tissues are placed into molds with the liquid embedding material, usually a water-based glycol, OCT, TBS, Cryogel, or resin, which is then frozen to form hardened blocks.

Sectioning Edit

For light microscopy, a knife mounted in a microtome is used to cut tissue sections (typically between 5-15 micrometers thick) which are mounted on a glass microscope slide. [9] For transmission electron microscopy (TEM), a diamond or glass knife mounted in an ultramicrotome is used to cut between 50-150 nanometer thick tissue sections. [9]

Staining Edit

Biological tissue has little inherent contrast in either the light or electron microscope. [17] Staining is employed to give both contrast to the tissue as well as highlighting particular features of interest. When the stain is used to target a specific chemical component of the tissue (and not the general structure), the term histochemistry is used. [9]

Light microscopy Edit

Hematoxylin and eosin (H&E stain) is one of the most commonly used stains in histology to show the general structure of the tissue. [9] [18] Hematoxylin stains cell nuclei blue eosin, an acidic dye, stains the cytoplasm and other tissues in different stains of pink. [9] [12]

In contrast to H&E, which is used as a general stain, there are many techniques that more selectively stain cells, cellular components, and specific substances. [12] A commonly performed histochemical technique that targets a specific chemical is the Perls' Prussian blue reaction, used to demonstrate iron deposits [12] in diseases like hemochromatosis. The Nissl method for Nissl substance and Golgi's method (and related silver stains) are useful in identifying neurons are other examples of more specific stains. [12]

Historadiography Edit

In historadiography, a slide (sometimes stained histochemically) is X-rayed. More commonly, autoradiography is used in visualizing the locations to which a radioactive substance has been transported within the body, such as cells in S phase (undergoing DNA replication) which incorporate tritiated thymidine, or sites to which radiolabeled nucleic acid probes bind in in situ hybridization. For autoradiography on a microscopic level, the slide is typically dipped into liquid nuclear tract emulsion, which dries to form the exposure film. Individual silver grains in the film are visualized with dark field microscopy.

Immunohistochemistry Edit

Recently, antibodies have been used to specifically visualize proteins, carbohydrates, and lipids. This process is called immunohistochemistry, or when the stain is a fluorescent molecule, immunofluorescence. This technique has greatly increased the ability to identify categories of cells under a microscope. Other advanced techniques, such as nonradioactive in situ hybridization, can be combined with immunochemistry to identify specific DNA or RNA molecules with fluorescent probes or tags that can be used for immunofluorescence and enzyme-linked fluorescence amplification (especially alkaline phosphatase and tyramide signal amplification). Fluorescence microscopy and confocal microscopy are used to detect fluorescent signals with good intracellular detail.

Electron microscopy Edit

For electron microscopy heavy metals are typically used to stain tissue sections. [9] Uranyl acetate and lead citrate are commonly used to impart contrast to tissue in the electron microscope. [9]

Specialized techniques Edit

Cryosectioning Edit

Similar to the frozen section procedure employed in medicine, cryosectioning is a method to rapidly freeze, cut, and mount sections of tissue for histology. The tissue is usually sectioned on a cryostat or freezing microtome. [12] The frozen sections are mounted on a glass slide and may be stained to enhance the contrast between different tissues. Unfixed frozen sections can be used for studies requiring enzyme localization in tissues and cells. Tissue fixation is required for certain procedures such as antibody-linked immunofluorescence staining. Frozen sections are often prepared during surgical removal of tumors to allow rapid identification of tumor margins, as in Mohs surgery, or determination of tumor malignancy, when a tumor is discovered incidentally during surgery.

Ultramicrotomy Edit

Ultramicrotomy is a method of preparing extremely thin sections for transmission electron microscope (TEM) analysis. Tissues are commonly embedded in epoxy or other plastic resin. [9] Very thin sections (less than 0.1 micrometer in thickness) are cut using diamond or glass knives on an ultramicrotome. [12]

Artifacts Edit

Artifacts are structures or features in tissue that interfere with normal histological examination. Artifacts interfere with histology by changing the tissues appearance and hiding structures. Tissue processing artifacts can include pigments formed by fixatives, [12] shrinkage, washing out of cellular components, color changes in different tissues types and alterations of the structures in the tissue. An example is mercury pigment left behind after using Zenker's fixative to fix a section. [12] Formalin fixation can also leave a brown to black pigment under acidic conditions. [12]

In the 17th century the Italian Marcello Malpighi used microscopes to study tiny biological entities some regard him as the founder of the fields of histology and microscopic pathology. [19] [20] Malpighi analyzed several parts of the organs of bats, frogs and other animals under the microscope. While studying the structure of the lung, Malpighi noticed its membranous alveoli and the hair-like connections between veins and arteries, which he named capillaries. His discovery established how the oxygen breathed in enters the blood stream and serves the body. [21]

In the 19th century histology was an academic discipline in its own right. The French anatomist Xavier Bichat introduced the concept of tissue in anatomy in 1801, [22] and the term "histology" (German: Histologie), coined to denote the "study of tissues", first appeared in a book by Karl Meyer in 1819. [23] [24] [19] Bichat described twenty-one human tissues, which can be subsumed under the four categories currently accepted by histologists. [25] The usage of illustrations in histology, deemed as useless by Bichat, was promoted by Jean Cruveilhier. [26] [ when? ]

In the early 1830s Purkynĕ invented a microtome with high precision. [24]

Mounting techniques were developed by Rudolf Heidenhain (1824-1898), who introduced gum Arabic Salomon Stricker (1834-1898), who advocated a mixture of wax and oil and Andrew Pritchard (1804-1884) who, in 1832, used a gum/isinglass mixture. In the same year, Canada balsam appeared on the scene, and in 1869 Edwin Klebs (1834-1913) reported that he had for some years embedded his specimens in paraffin. [27]

The 1906 Nobel Prize in Physiology or Medicine was awarded to histologists Camillo Golgi and Santiago Ramon y Cajal. They had conflicting interpretations of the neural structure of the brain based on differing interpretations of the same images. Ramón y Cajal won the prize for his correct theory, and Golgi for the silver-staining technique that he invented to make it possible. [28]

In vivo histology Edit

Currently there is intense interest in developing techniques for in vivo histology (predominantly using MRI), which would enable doctors to non-invasively gather information about healthy and diseased tissues in living patients, rather than from fixed tissue samples. [29] [30] [31] [32]


What is the workplace of a Biologist like?

Most biologists are employed by governmental agencies, universities, or private industry laboratories. Many biologists at universities are also professors, and spend most of their time teaching students research methods, assisting with the development of the students' projects, as well as working on their own projects.

Biological scientists employed by private industries and by the government are able to focus more on their own personal projects and those assigned by their superiors. Some examples of biologists likely to be working in private industries are zoologists and ecologists, who could be employed by zoos and environmental agencies.

The area of biology that one is employed in will determine if more time will be spent in the laboratory or outside in the field. Histotechnologists, for example, work in a laboratory environment, as their work involves preparing tissues for microscopic examination. Botanists, ecologists, and zoologists, on the other hand, spend a lot of their time in the field, studying plants and animals in various climates and habitats while often living in primitive conditions.

In general, most biological scientists do not experience much in the way of dangerous situations. Those studying dangerous or toxic organisms have a series of special precautions they take to prevent contamination and any possibility of spreading viruses or bacteria.


Historical background

Theophrastus, a Greek philosopher who first studied with Plato and then became a disciple of Aristotle, is credited with founding botany. Only two of an estimated 200 botanical treatises written by him are known to science: originally written in Greek about 300 bce , they have survived in the form of Latin manuscripts, De causis plantarum and De historia plantarum. His basic concepts of morphology, classification, and the natural history of plants, accepted without question for many centuries, are now of interest primarily because of Theophrastus’s independent and philosophical viewpoint.

Pedanius Dioscorides, a Greek botanist of the 1st century ce , was the most important botanical writer after Theophrastus. In his major work, an herbal in Greek, he described some 600 kinds of plants, with comments on their habit of growth and form as well as on their medicinal properties. Unlike Theophrastus, who classified plants as trees, shrubs, and herbs, Dioscorides grouped his plants under three headings: as aromatic, culinary, and medicinal. His herbal, unique in that it was the first treatment of medicinal plants to be illustrated, remained for about 15 centuries the last word on medical botany in Europe.

From the 2nd century bce to the 1st century ce , a succession of Roman writers—Cato the Elder, Varro, Virgil, and Columella—prepared Latin manuscripts on farming, gardening, and fruit growing but showed little evidence of the spirit of scientific inquiry for its own sake that was so characteristic of Theophrastus. In the 1st century ce , Pliny the Elder, though no more original than his Roman predecessors, seemed more industrious as a compiler. His Historia naturalis—an encyclopaedia of 37 volumes, compiled from some 2,000 works representing 146 Roman and 327 Greek authors—has 16 volumes devoted to plants. Although uncritical and containing much misinformation, this work contains much information otherwise unavailable, since most of the volumes to which he referred have been destroyed.

The printing press revolutionized the availability of all types of literature, including that of plants. In the 15th and 16th centuries, many herbals were published with the purpose of describing plants useful in medicine. Written by physicians and medically oriented botanists, the earliest herbals were based largely on the work of Dioscorides and to a lesser extent on Theophrastus, but gradually they became the product of original observation. The increasing objectivity and originality of herbals through the decades is clearly reflected in the improved quality of the woodcuts prepared to illustrate these books.

In 1552 an illustrated manuscript on Mexican plants, written in Aztec, was translated into Latin by Badianus other similar manuscripts known to have existed seem to have disappeared. Whereas herbals in China date back much further than those in Europe, they have become known only recently and so have contributed little to the progress of Western botany.

The invention of the optical lens during the 16th century and the development of the compound microscope about 1590 opened an era of rich discovery about plants prior to that time, all observations by necessity had been made with the unaided eye. The botanists of the 17th century turned away from the earlier emphasis on medical botany and began to describe all plants, including the many new ones that were being introduced in large numbers from Asia, Africa, and America. Among the most prominent botanists of this era was Gaspard Bauhin, who for the first time developed, in a tentative way, many botanical concepts still held as valid.

In 1665 Robert Hooke published, under the title Micrographia, the results of his microscopic observations on several plant tissues. He is remembered as the coiner of the word “cell,” referring to the cavities he observed in thin slices of cork his observation that living cells contain sap and other materials too often has been forgotten. In the following decade, Nehemiah Grew and Marcello Malpighi founded plant anatomy in 1671 they communicated the results of microscopic studies simultaneously to the Royal Society of London, and both later published major treatises.

Experimental plant physiology began with the brilliant work of Stephen Hales, who published his observations on the movements of water in plants under the title Vegetable Staticks (1727). His conclusions on the mechanics of water transpiration in plants are still valid, as is his discovery—at the time a startling one—that air contributes something to the materials produced by plants. In 1774, Joseph Priestley showed that plants exposed to sunlight give off oxygen, and Jan Ingenhousz demonstrated, in 1779, that plants in the dark give off carbon dioxide. In 1804 Nicolas de Saussure demonstrated convincingly that plants in sunlight absorb water and carbon dioxide and increase in weight, as had been reported by Hales nearly a century earlier.

The widespread use of the microscope by plant morphologists provided a turning point in the 18th century—botany became largely a laboratory science. Until the invention of simple lenses and the compound microscope, the recognition and classification of plants were, for the most part, based on such large morphological aspects of the plant as size, shape, and external structure of leaves, roots, and stems. Such information was also supplemented by observations on more subjective qualities of plants, such as edibility and medicinal uses.

In 1753 Linnaeus published his master work, Species Plantarum, which contains careful descriptions of 6,000 species of plants from all of the parts of the world known at the time. In this work, which is still the basic reference work for modern plant taxonomy, Linnaeus established the practice of binomial nomenclature—that is, the denomination of each kind of plant by two words, the genus name and the specific name, as Rosa canina, the dog rose. Binomial nomenclature had been introduced much earlier by some of the herbalists, but it was not generally accepted most botanists continued to use cumbersome formal descriptions, consisting of many words, to name a plant. Linnaeus for the first time put the contemporary knowledge of plants into an orderly system, with full acknowledgment to past authors, and produced a nomenclatural methodology so useful that it has not been greatly improved upon. Linnaeus also introduced a “sexual system” of plants, by which the numbers of flower parts—especially stamens, which produce male sex cells, and styles, which are prolongations of plant ovaries that receive pollen grains—became useful tools for easy identification of plants. This simple system, though effective, had many imperfections. Other classification systems, in which as many characters as possible were considered in order to determine the degree of relationship, were developed by other botanists indeed, some appeared before the time of Linnaeus. The application of the concepts of Charles Darwin (on evolution) and Gregor Mendel (on genetics) to plant taxonomy has provided insights into the process of evolution and the production of new species.

Systematic botany now uses information and techniques from all the subdisciplines of botany, incorporating them into one body of knowledge. Phytogeography (the biogeography of plants), plant ecology, population genetics, and various techniques applicable to cells—cytotaxonomy and cytogenetics—have contributed greatly to the current status of systematic botany and have to some degree become part of it. More recently, phytochemistry, computerized statistics, and fine-structure morphology have been added to the activities of systematic botany.

The 20th century saw an enormous increase in the rate of growth of research in botany and the results derived therefrom. The combination of more botanists, better facilities, and new technologies, all with the benefit of experience from the past, resulted in a series of new discoveries, new concepts, and new fields of botanical endeavour. Some important examples are mentioned below.

New and more precise information is being accumulated concerning the process of photosynthesis, especially with reference to energy-transfer mechanisms.

The discovery of the pigment phytochrome, which constitutes a previously unknown light-detecting system in plants, has greatly increased knowledge of the influence of both internal and external environment on the germination of seeds and the time of flowering.

Several types of plant hormones (internal regulatory substances) have been discovered—among them auxin, gibberellin, and kinetin—whose interactions provide a new concept of the way in which the plant functions as a unit.

The discovery that plants need certain trace elements usually found in the soil has made it possible to cultivate areas lacking some essential element by adding it to the deficient soil.

The development of genetical methods for the control of plant heredity has made possible the generation of improved and enormously productive crop plants.

The development of radioactive-carbon dating of plant materials as old as 50,000 years is useful to the paleobotanist, the ecologist, the archaeologist, and especially to the climatologist, who now has a better basis on which to predict climates of future centuries.

The discovery of alga-like and bacteria-like fossils in Precambrian rocks has pushed the estimated origin of plants on Earth to 3,500,000,000 years ago.

The isolation of antibiotic substances from fungi and bacteria-like organisms has provided control over many bacterial diseases and has contributed biochemical information of basic scientific importance as well.

The use of phylogenetic data to establish a consensus on the taxonomy and evolutionary lineages of angiosperms (flowering plants) is coordinated through an international effort known as the Angiosperm Phylogeny Group.


Branches of Biology

Zoology

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This is a branch of biology that studies animals. The term zoology originated from the Greek term “Zoon” meaning animal and “logos” meaning study. Zoology is divided into Applied Zoology, the study of production and non production animals, Systematic Zoology, dealing with evolution and taxonomy or science of naming living things and Organismal Zoology, the study of animals in our biosphere. Applied Zoology is further divided into, Aquaculture, which involves production and maintenance of freshwater and seawater animals and plants, Piggery, which includes study of everything related to pigs, Applied Entomology,which includes manipulation of insects for the benefit of humans, Vermiculture, which is breeding of the worms which burrow soil, for production of natural fertilizers, Poultry Science, the study of domestic birds such as geese, turkey and chicken, Parasitology, dealing with the study of parasites, Radiation Biology, which uses gamma rays, X-rays, electrons and protons for well-being of humans, Biotechnology, which applies engineering principles for the material processing by biological factors, Applied Embryology, which embraces test tube culture (embryo culture) for increasing productivity from cattle, Tissue Culture, involving the culture of plant tissues and cells in an artificial environment, Dairy Science, which deals with milk or milk related products, Pesticide Technology, which is the study of pesticides and their uses, Nematology which deals with study of roundworms of organisms and their control, Ornithology, which is the study of birds, Herpetology, study of reptiles, Ichthyology, which is the study of fish and Mammology, which includes the study of mammals.

Entomology

One of the sub branches is entomology, which is exclusively based on insects. It concentrates on studying the taxonomy, features, adaptations, roles and behavior of insects.

Ethology

Truly speaking, ethology comes under zoology and deals with behavioral adaptations of animals, specially in their natural or original dwelling places.

Anatomy

Applicable to plant anatomy and animal anatomy, it involves studying the detailed structure, internal organs and the respective functions of an organism.

Physiology

Physiology is defined as the study of various functions and processes of living organisms. Physiology is further divided into Evolutionary Physiology, which is the study of physiological evolution, Cell Physiology – the study of cell mechanism and interaction, Developmental Physiology, which involves the study of physiological processes in relation to embryonic evolution, Environmental Physiology, which deals with the study of response of plants to agents such as temperature, radiation and fire and Comparative Physiology, roughly explained as the study of animals except humans.

Genetics

This is considered to be an interesting field of study and is a branch of biology. Genetics is the study of genes. This term is derived from the Greek word “genetikos” meaning “origin”. This branch of biology studies about the hereditary aspects of all living organisms. The study of inheritance of traits from the parent had begun in the mid-nineteenth century and was pioneered by a renowned biologist Gregor Mendel. The modern science of genetics is based upon the foundations laid down by this biologist.

Botany

The study of plant life or phytology is known as botany. One of the most prominent among the different branches of biology, botany is a vast subject and studies the life and development of fungi, algae and plants. Botany also probes into the structure, growth, diseases, chemical and physical properties, metabolism and evolution of the plant species. Botany implies the importance of study of plant life on earth because they generate food, fibers, medicines, fuel and oxygen.

Evolution Biology

As we all know, highly developed organism have evolved from simpler forms. There is a specific branch of biology, called evolution biology that focuses on the evolution of species.

Developmental Biology

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As the name signifies, development biology helps a student in learning the various phases of growth and development of a living creature.

Ecology

Ecology is a branch of biology that studies the interaction of various organisms with one another, and their chemical and physical environment. This branch of biology studies environmental problems such as pollution and how it affects the eco-cycle. The term ecology is derived from the Greek term “oikos” meaning “household” and “logos” meaning “study”. A German biologist, Ernst Haeckel, coined the term ecology in 1866.

Cryobiology

This deals with the effects of extremely low temperature in living cells and organisms as a whole.

Biochemistry

This branch of biology studies the chemical processes in all living organisms. Biochemistry is a branch of science that studies the functions of the cellular components such as nucleic acids, lipids, proteins and various other bio-molecules.

Cytology and Molecular Biology

In-depth study about the cell along with its structure, function, parts and abnormalities are all studied under cell biology or cytology. Likewise, study of organisms at the molecular level is called molecular biology.

Marine Biology

Marine biology studies the ecosystem of the oceans, marine animals and plants. There is a vast portion of ocean life that is still unexplored. You can rightly say that marine biology is a branch of oceanography, which is, again, a branch of biology.

Bioinformatics

Bioinformatics basically relates to genomic studies with the application of data processing, computational knowledge and statistical applications.

Mycology

According to modern-day taxonomy, fungi (singular fungus) is neither a plant nor an animal. It belongs to a different living group and is studied under the subject, mycology.

Biophysics

Biophysics involves the study of relation between organisms or living cells and electrical or mechanical energy. Biophysics is further divided into the following sub-branches: Molecular Biophysics, which defines biological functions in relation to dynamic behavior and molecular structure of various living systems such as viruses, Bio mechanics is the study of forces applied by muscles and gravity on the skeleton, Bio electricity – the study of electric currents flowing through muscles and nerves and static voltage of biological cells, Cellular Biophysics, which incorporates study of membrane function and structure, and cellular excitation and Quantum Biophysics, which includes the study of behavior of living matter at molecular and sub molecular level.

Aquatic Biology

It involves study of life in water, like study of various species of animals, plants and micro-organisms. It incorporates the study of both freshwater and sea water organisms. Sometimes, aquatic biology is also referred to as limnology.

Biology as a science gives us the opportunity to make observations, evaluate and solve problems that are related to plants and animals. If you are interested in biology, pursuing a career in any branch of biology can be immensely rewarding.

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Read on to know more about the steps of photosynthesis, one of nature's most fascinating occurrence.


G. state whether the following statements are true or false if false rewrite the correct form of statement:

  1. Osmosis and diffusion are same phenomena .
    • False, Osmosis is a special type of diffusion which require the presence of semi permeable membrane, that allows the movement of solvent only.
  2. Plants lose water by the process called translocation.
    • False, Plants lose water by the process called transpiration .
  3. Root hairs are multicellular structures.
    • False, root hairs are unicellular structures.
  4. Xylem and phloem are vascular tissues .
    • True.
  5. Water transport in unicellular plants takes place by diffusion.
    • True.

Results

Patterning of Root Tissues Is Determined by the Local Availability of Water.

Soil is a heterogeneous environment containing particles and aggregated structures of different sizes with pockets of air and nonuniform distributions of water and nutrients (6). Our understanding of how roots sense and interpret microscale heterogeneity is poor due, in part, to a lack of model experimental systems for studying such phenomena. Interestingly, growing Arabidopsis seedling roots along the surface of an agar medium creates spatial asymmetries in the environment the primary root is exposed to, but the effect of these differences has not been explored before. Under these conditions, one side of the primary root is in contact with the agar medium and the film of water that forms on its surface (contact side), whereas the other side of the primary root is exposed to air in the headspace of the Petri dish (air side) (Fig. 1A). We found that seedlings grown on agar media infrequently developed LRs that grew straight out into the air, suggesting that their patterning might be influenced by this environmental asymmetry (Fig. 1 B, C, and D). We created a phenotyping key to quantify the emergence patterns of LRs across the circumferential axis by categorizing emerged LRs as air side, horizon side, or contact side (SI Appendix, SI Materials and Methods, provides a full description of criteria used). Although we would have a priori expected that the chance of an LR emerging from any particular side of the primary root would be nearly equivalent, we instead observed a strong bias in LR emergence toward the contact side (Fig. 1D). This bias was lost when roots were grown through agar (Fig. 1 A and D), indicating that the observed phenomenon requires an asymmetric environment. Root curvature influences at which side of the root (concave or convex) a LR will initiate (3). We found that inducing a 90-degree bend in the root through gravitropic stimulation had no significant influence on the bias in LR development that occurred along the air–agar axis suggesting that these two processes act independently on the distribution of LRs (SI Appendix, Fig. S1).

Hydropatterning of root development in Arabidopsis, maize and rice. (A) Diagram showing asymmetries in the local environment generated when seedlings grow on the surface of an agar-based media or the symmetric environment generated when roots are grown through agar. (B and C) LR primordia emerging from the contact (B) or air side (C) of the primary root. (D) Quantification of LR emergence patterns from the primary root under different conditions (n > 10). Various phenotypic categories are indicated with different colors and are marked in A. (E) Cross-section of a rice primary root grown on agar, stained with calcofluor. Image shows the development of aerenchyma (AE) and root hairs (RH) on the air side and an LR emerging from the contact side. (F) Cross-section of a maize root grown on agar and stained with propidium iodide. (G) Diagram showing the construction of “agar sandwiches” used to test the effects of local differences in media composition on LR development in maize. (H) LR outgrowth is induced on two sides by contact with agar (Mock/Control) this effect is diminished on the “Treatment” side when the water potential of the media is reduced using PEG infusion (PEG/Control). Contact of the root with a glass surface does not induce LR outgrowth (Glass/Control). Growth of roots along a single agar surface results in the suppression of LR development on the air side (Air/Control) (n > 10). (IK) MicroCT-generated images of maize seedlings grown through a macropore of air (I and K) or a continuous volume of soil (J). The root in K is growing in air, whereas in I the root is contacting the soil surface. Root tissue is false-colored in white, and soil is false-colored in brown. Average number of LRs per seedling (D) and per centimeter of primary root (H) is shown at base of columns in bar charts. Error bars indicate SEM. Significant differences based on Fisher’s exact test (P < 0.05) with similar groups are indicated using the same letter.

Changing agar concentration in the growth media from 1 to 2% decreased water potential (Ψw) and the amount of expressed water on the surface of the media, which reduced the circumferential area of the root contacting liquid water (SI Appendix, Fig. S1) (7). This change in media composition also significantly affected the bias in the distribution of LRs (Fig. 1D). Use of different growth substrates indicated that no specific component of the media besides water was necessary to elicit biased LR development, although these media differed significantly in water potential (range, −0.22 MPa) (SI Appendix, Fig. S2). These data suggest that contact with water, in or on the surface of the media, had a greater influence on the local induction of LR development than small differences in water potential.

Growth of Oryza sativa (rice) and Zea mays (maize) seedlings on agar also resulted in the development of LRs predominantly on the contact side of the root (Fig. 1 E, F, and H and SI Appendix, Fig. S3). In maize, LR development was also locally induced when seedlings were grown on wet germination paper, again indicating that no specific component of the medium besides water was necessary to elicit biased LR development (SI Appendix, Fig. S3). Using a similar experimental system as Karahara et al. (8) (Fig. 1G), we tested the effects of placing the maize primary root between two slabs of control media. Interestingly, LRs developed along both sides contacting the media, demonstrating that multiple distinct domains along the circumferential axis of the root can simultaneously form LRs with intervening areas lacking LR development (Fig. 1H).

X-ray microscale computed tomography (microCT) visualization of maize roots growing through a macropore (large air space) in the soil matrix revealed a similar positioning of LRs biased toward the root face in direct contact with the soil (Fig. 1I, Movie S1, and SI Appendix, Table S1). When roots were grown in pots without a macropore, LRs developed around the entire circumference of the primary root (Fig. 1J and Movie S2). Interestingly, when roots did not contact the soil surface in the macropore (Fig. 1K and Movie S3), LRs emerged sporadically in all directions, suggesting that a nonuniform environment is required for the bias in LR development but that contact is not required for LR development per se in this condition. These data support the physiological relevance of the patterning phenomenon observed in vitro.

In addition to LR emergence, rice and maize seedlings showed preferential accumulation of aerenchyma (air pockets forming in the cortex cell layers that may aid in gas exchange) on the air side of the root (Fig. 1 E and F and SI Appendix, Fig. S3). In maize, the pigment anthocyanin accumulated on the air side of the root whereas it was depleted from the contact side, especially in those regions where preemergent LRs were developing (SI Appendix, Fig. S3). This provided a useful visual marker to distinguish contact and air sides in root cross-sections. Anthocyanin biosynthesis is light-dependent however, hydropatterning of LRs was not disrupted by growth of plants in the dark (SI Appendix, Fig. S3).

Rice, maize, and Arabidopsis also showed a clear bias in root hair development on the air side (Fig. 1 E and F and SI Appendix, Fig. S4). In Arabidopsis, suppression of root hair development often occurred before the initiation stage but was not associated with obvious changes in the expression of genes involved in root hair patterning (SI Appendix, Fig. S4). The presence of root hairs was used as a visual marker to distinguish air and contact sides of roots removed from the media for imaging. Root hair initiation on the contact side could be rescued by treatment with abscisic acid (ABA) or the ethylene precursor 1–aminocyclopropane-1–carboxylic acid (ACC), suggesting that the lack of root hair development was not simply a consequence of physical impedance of the growth medium (SI Appendix, Fig. S4). Together these data demonstrated that plant roots are adept at sensing and developmentally responding to local differences in the environment in ways that we hypothesize take advantage of microscopic variations in the distribution of liquid water and air in soil.

The rate at which water is absorbed by the root (Jv) is the product of the driving force for water flow (ΔΨw, the difference in water potential between the root and the growth medium) and the resistance to water flow (inversely proportional to the hydraulic conductivities of the medium and the root, Lp) (9). In our in vitro growth systems, the air is likely at water-potential equilibrium with the culture medium thus water potential does not distinguish these environments. Hydraulic conductivity, however, differs dramatically the conductivity of agar (1 × 10 −5 m 2 s −1 MPa −1 ) is orders of magnitude higher than that of air (4.18 × 10 −12 m 2 s −1 MPa −1 ) (9, 10).

To specifically test the effects that media water potential and hydraulic conductivity have on the local regulation of LR development, we again used the “agar sandwich” approach to vary the media contacting the maize root (treatment agar slab) while a second agar slab contacting the root served as a control. In rice, Karahara et al. (8) previously showed that growth of roots between two slabs of agar results in asymmetries in aerenchyma development if one of the slabs contains mannitol, which reduces water potential of the medium. We performed similar experiments using polyethylene glycol (PEG infused agar Ψw was −0.63 ± 0.02 MPa, and control agar was −0.10 ± 0.01 MPa) and observed a significant reduction in LR emergence on the treatment side, which partially mimicked the effect of air (Fig. 1H) (11). We severely reduced hydraulic conductivity by placing various non-water-conducting materials between the root and the treatment agar slab. This eliminated the inductive effect of this media on LR development, indicating that hydraulic conductivity of the contacted surface, rather than contact alone, was important for hydropatterning (SI Appendix, Fig. S3). Similar results were obtained when a sheet of glass or silicone rubber was used to contact the root, suggesting that the pliancy of the material was inconsequential (Fig. 1H and SI Appendix, Fig. S3). Together these data suggest that the rate with which water is absorbed by a root from the media determines whether a contacted surface will induce LR development.

To describe the environmental response phenomena shown here, we have designated the term hydropatterning: a nonuniform distribution of available water causes asymmetries in root development. This term is primarily used to simplify discussion of the process, and we do not intend to imply any specific physiological or molecular mechanisms used by the plant to detect differences in water availability.

Hydropatterning Affects Lateral Root Development During FC Specification.

In Arabidopsis, the developmental steps involved in LR patterning have been well defined (12). Previous studies have clearly shown that environmental stimuli can affect the initiation and emergence of LRs (1) however, evidence is lacking regarding an earlier role. We predicted that if hydropatterning acts after LR initiation we should observe an accumulation of preemerged LR primordia on the air side, which would account for the lower relative number of emerged LRs on this side.

The ProMiR390a:GFP-GUS reporter is expressed in the xylem pole and associated pericycle cells and marks stage-I LR primordia and later stages (13) (Fig. 2A). Confocal imaging of contact and air sides of seedling primary roots showed a clear bias in the number of GFP-positive foci observed between these sides (Fig. 2A). The ProDR5:VENUS-N7 reporter is initially expressed in adjacent xylem pole pericycle cells during FC activation and can be used to visualize the migration of nuclei from two neighboring pericycle cells to the common anticlinal cell plate before cell division and stage-I LR initiation (Fig. 2B) (14, 15). Interestingly, several stages of LR development showed a bias between the contact and air sides of the root, and no obvious accumulation of paused or quiescent LR primordia was observed on the air side that could account for the difference in emerged primordia between these sides (Fig. 2B). Quantification of LR primordia in seedlings after tissue clearing revealed similar results (SI Appendix, Fig. S5). These data indicate that hydropatterning acts at or before the earliest stages of LR initiation.

Hydropatterning acts during FC specification to affect LR patterning. (A) The PromiR390a:GUS-GFP reporter is expressed at stage 1 of LR initiation and later. Confocal imaging of contact and air sides of the primary root showed a strong bias in the number of GFP-positive foci (n ≥ 10). (B) The ProDR5:N7:VENUS reporter marks pericycle cell nuclei at the stage of FC activation (FCA), Stage 1 (S1), Stage 2 (S2), and later stages. Most stages showed greater numbers of primordia on the contact side than on the air side. (C) Seedling expressing the ProDR5:LUC+ reporter. LR emergence patterns were quantified in the region of the primary root containing all emerged LRs (in this example, the region above the yellow arrow). Luciferase activity was then visualized, and foci of reporter activity, included outgrown LRs, were counted (n = 27). Quiescent PBSs (red arrows) are sites of reporter expression that showed no signs of LR emergence. (D) Chart showing the number of emerged LRs and quiescent PBSs (QPBS). (E) Seedlings were grown for 5 d on 1% agar, then a second agar slab was applied to the former air side of the root. Seedlings were grown for five additional days, and the position of emerged LRs was quantified in the region of the primary root that formed before and after treatment. Control seedlings were grown similarly however, a second agar slab was not applied. The proximal domain is defined as the region of the primary root in contact with the second applied agar slab, whereas the distal domain is toward the original agar slab on which seedlings were germinated. In B, significant differences were analyzed on a per-stage basis using Student’s t test (P < 0.05) statistically similar groups are indicated using the same letter. For E, asterisk indicates a significant difference based on Fisher’s exact test (P < 0.05). Average number of LRs per centimeter of primary root shown at base of columns in bar charts. Error bars indicate SEM. (Scale bar, 50 μm.)

The orientation of the xylem pole determines the angle with which LRs emerge (5). Using the ProS32:erGFP reporter to mark the orientation of the vascular pole we found no significant bias with respect to the air–agar axis, eliminating this as a possible contributor to hydropatterning (SI Appendix, Fig. S5).

The earliest visual marker for the position of future LR primordia is the ProDR5:LUC+ reporter, which marks PBSs. Moreno-Risueño et al. (4) previously showed that growth of roots on the surface or through agar had no significant influence on the number of PBSs specified, indicating that hydropatterning acts subsequent to PBS specification. Between the specification of PBSs along the longitudinal axis and the activation of asymmetric divisions in FCs, an additional decision must be made that has received less attention. In Arabidopsis, LRs will only develop from pericycle cells that overlie one of the two xylem poles (5). Although two such populations of cells exist along the circumferential axis of the root, pericycle cells adjacent to only one xylem pole are chosen. We postulated two models for how hydropatterning affects LR patterning during this developmental interval (SI Appendix, Fig. S6). In the first model, xylem pole selection and the subsequent specification of FCs at this pole are independent of the local environment, with the later stage of LR initiation being the target of hydropatterning. This model predicts that a similar number of FCs should be specified on the air and contact sides, with most FCs on the air side remaining quiescent. The second model postulates that local environmental differences across the circumferential axis bias xylem pole selection and FC specification toward the contact side. Based on this second model, we would expect few quiescent PBSs as most would be specified toward the permissive environment of the contacted surface to begin with.

Under our growth conditions, seedlings expressing the ProDR5:LUC+ reporter developed on average 11.9 total emerged LRs after 10 d of growth with 7.4 LRs emerged toward the agar and 1.2 toward the air (Fig. 2 C and D). Thus, the difference in emerged LRs between air and contact sides is ∼6.3. To determine if there existed a quantity of quiescent PBSs that would account for this difference, we visualized the ProDR5:LUC+ reporter and found 13.4 LUC-marked sites. We verified that reporter expression in PBSs was maintained throughout the time frame of the experiment (SI Appendix, Fig. S7). Based on the difference between the number of LUC-expressing foci and emerged LRs, we calculated that 1.5 PBSs were quiescent per seedling. This number is significantly lower than the number expected based on the first model (6.3 quiescent PBSs). Thus, our data are consistent with the second model and suggest that hydropatterning likely acts during FC specification.

If hydropatterning acts at FC specification, then mature regions of the primary root where FCs have already been specified should not be responsive to future changes in the distribution of water in the environment. By this reasoning, we predicted that regions of the root previously exposed to air would not develop new LRs if they subsequently came into contact with a wet surface. We directly tested this prediction by applying a sheet of agar to the air side of the root of an Arabidopsis seedling 5 d postgermination. The orientation of LR emergence was quantified in regions of the primary root that had formed before and after the application of the agar slab. In regions of the primary root previously exposed to air, the spatial distribution of LRs was similar in the treated roots to the untreated control, whereas in regions of the primary root that formed after application of the agar slab, LRs developed in all directions (Fig. 2E). These results are consistent with our model that hydropatterning acts at the time of FC specification and suggest that the orientation of LRs is determined through sensing of the local environment near the root tip and subsequently becomes fixed.

Auxin Biosynthesis Is Moisture-Induced and Necessary for Hydropatterning.

We next asked which signaling pathways act downstream of moisture during hydropatterning. Previous work has shown that under water-limiting conditions, LR development is strongly suppressed (16). Severe water limitation induces ABA signaling, which is known to inhibit the development of LRs (17). We found that several mutants that disrupt ABA signaling did not have a significant effect on hydropatterning (SI Appendix, Fig. S8). Of particular importance, the pyr/pyl 112458 mutant, which is highly resistant to ABA treatment (18), showed normal hydropatterning (SI Appendix, Fig. S8). These data differentiate hydropatterning from a classic water-stress response and indicate that signaling pathways other than ABA are involved.

Auxin is an important signaling molecule contributing to all stages of LR development (19). Quantification of indole-3–acetic acid (IAA) concentration in whole roots of Arabidopsis showed a significant increase when seedlings were grown on media with a lower concentration of agar (Fig. 3A). Measurements of endogenous auxin signaling using the DII-VENUS sensor, which is degraded in an auxin concentration-dependent manner (20), showed similar results (SI Appendix, Fig. S9). Furthermore, a transcriptional reporter of auxin response, ProDR5:erGFP, showed an increase in fluorescence in the outer tissue layers of the root at 1% agar compared with 3% agar conditions (SI Appendix, Fig. S9). These results suggest that water availability promotes auxin accumulation, signaling, and response.

Moisture activates auxin biosynthesis and response. (A) IAA levels quantified by liquid chromatography tandem mass spectrometry in whole roots grown on media containing different concentrations of agar (n = 3). (B) Maximum projects of confocal image stacks show DII-VENUS reporter expression is higher on the air side of the root relative to the contact side. Fluorescence intensity is shown using a 16-color look-up table. (C) Quantitation of DII-VENUS average nuclear fluorescence intensity in the epidermis shown for different regions of the root. (D) Cross-sections of rice roots expressing the ProDR5:GUS reporter showing local induction of the reporter on the contact side of the root (on agar) and uniform activation of the reporter when roots are grown in agar. (E and F) The ProTIR2:TIR2:GUS reporter shows stronger expression in the outer tissue layers of seedlings grown on 1% compared with 3% agar, quantified in F. (G) Two mutant alleles of tryptophan aminotransferase of Arabidopsis (TAA1) show a strong suppression of hydropatterning (n ≥ 20). (H) Maximum projections of confocal image stacks show ProTAA1:GFP:TAA1 reporter expression in the LRC and epidermis of the transition and elongation zones. (I) GFP fluorescence quantified for cell types on the air and contact sides (n ≥ 8). (J) The ProWER:TAA1 transgene was able to rescue the wei8-1 hydropatterning defect in multiple independent transgenic lines as was growth of seedlings on media supplemented with IAA (K). Average number of LRs per seedling shown at base of columns in bar charts. Error bars indicate SEM. Significant differences based on Fisher’s exact test (P < 0.05) (G, J, and K) or Student’s t test (P < 0.05) (A, C, F, and I) with similar groups indicated using same letter. (Scale bars, 50 μm.)

We asked whether the auxin pathway was locally regulated by contact with a wet surface. Expression of the DII-VENUS sensor was lower on the contact side of the early maturation zone, relative to the air side, although no significant differences were observed elsewhere (Fig. 3 B and C). These data suggest that higher levels of auxin may be present on the contact side of the primary root. Determining differences in auxin signaling at the elongation zone and pericycle cell layers was not possible using the DII-VENUS sensor, as fluorescence intensity was very low in these regions of the root. In rice, where radial cross-sections are more easily examined, the auxin transcriptional response reporter, ProDR5:GUS, exhibited higher staining on the contact side of the early maturation zone compared with the air side, whereas uniform activation of the reporter was observed in roots grown through agar (Fig. 3D). Together these data suggest that water availability can locally promote auxin accumulation and its response.

TAA1 (also known as WEI8, SAV3 and TIR2) encodes an l -tryptophan pyruvate aminotransferase that converts tryptophan to indole-pyruvic acid, a direct biosynthetic precursor of the auxin, IAA (21 ⇓ ⇓ ⇓ ⇓ –26). The ProTIR2:TIR2:GUS reporter showed an increase in expression in the outer tissue layers at 1% agar compared with 3% agar media, suggesting TAA1 may control water-dependent changes in auxin biosynthesis (Fig. 3 E and F). Indeed the ProTAA1:TAA1:GFP reporter line showed significant differences in expression level between the contact and air sides of the root (Fig. 3 H and I) for the lateral root cap (LRC) however, in the epidermis no differences were observed.

TAA1 loss-of-function alleles wei8-1 and sav3-1 both showed a significant reduction in hydropatterning (Fig. 3G), indicating that TAA1-mediated auxin biosynthesis is necessary for the response. To test if the spatial pattern of TAA1 expression is important for hydropatterning, we attempted to rescue wei8-1 defects by introducing transgenes that drove constitutive expression in the epidermis (ProWEREWOLF:TAA1) or throughout the root (ProUBIQUTIN10:TAA1). Both constructs were able to rescue hydropatterning in wei8-1 and did not cause obvious gain-of-function defects (Fig. 3J and SI Appendix, Fig. S9). Furthermore, exogenous IAA added to the media could similarly rescue wei8-1 defects (Fig. 3K). These data suggest that TAA1-mediated auxin biosynthesis is necessary for hydropatterning but that localized expression of TAA1 and local biosynthesis of IAA are not required to communicate positional information generated by the local environment.

Auxin Efflux and Response Pathways Are Necessary for Hydropatterning.

Polar auxin transport is required to generate localized gradients of auxin important for patterning the site of future lateral organs (27). We treated seedlings with various concentrations of a transportable form of auxin, IAA, or 2,4-dichlorophenoxyacetic acid (2,4-D), which is not efficiently effluxed from cells (Fig. 4 A and B) (28). Although IAA did not have a significant effect, 2,4-D could strongly disrupt hydropatterning even at low concentrations, suggesting that the ability of the root to transport auxin may be important for hydropatterning. We surveyed various genetic backgrounds affected in PIN-mediated auxin efflux and found that the Pro35S:PIN1 line strongly disrupted the process (SI Appendix, Fig. S10). Loss of pin-formed 3 (PIN3) function had a modest effect, which was enhanced in the pin2/3/7 mutant background (Fig. 4C) (29). The pin7 and the pin3/7 double mutant also showed significant defects, whereas other pin alleles did not affect hydropatterning (SI Appendix, Fig. S10). These data indicate that a normal auxin transport pathway is required for hydropatterning, and this can be disrupted through misexpression of, or loss-of-function mutations in, certain transport pathway members.

Auxin efflux transport pathways are necessary for hydropatterning. Effect of IAA (A) or 2,4-D (B) treatment on hydropatterning (n ≥ 20). (C) pin3-4 and pin2/3/7 mutants showed defects in hydropatterning (n ≥ 20). (D) Roots expressing the ProPIN3:PIN3:GFP and ProDR5:N7:VENUS reporters. Optical cross-sections at the cortex cell layer (Upper) or at the pericycle (Lower). Propidium iodide counter stain (magenta), PIN3:GFP (green, plasma membrane localized), and N7:VENUS (cyan, nuclear). (E) A radial cross-section reveals strong localization to the lateral cross-walls between cortex cells (yellow arrow). (F) Frequency with which early stages of LR development are observed on the air and contact sides of the primary root and whether these primordia are associated with PIN3:GFP expression in ground tissue (n = 9). (G) Transactivation of axr3-1 expression in the COR/END had the strongest effect in suppressing hydropatterning (n ≥ 20). Average number of LRs per seedling shown at base of columns in bar charts. Error bars indicate SEM. Significant differences based on Fisher’s exact test (P < 0.05) with similar groups indicated using same letter. (Scale bars, 50 μm.)

We examined the spatial localization pattern of the PIN3 protein using a GFP reporter (ProPIN3:PIN3:GFP) and found specific enrichment in cortex and endodermal cells (ground tissue) overlying early-stage LR primordia on the contact side (Fig. 4 D and E). PIN3:GFP was localized to all surfaces of these cells and was particularly enriched at the cross-wall between neighboring cortex cells overlying LR primordia (Fig. 4E). Few early-stage primordia developed on the air side, and these were generally associated with very weak or absent PIN3:GFP expression. Quantification of these results revealed that early stage-I LR primordia were preferentially associated with these patches of PIN3:GFP expression on the contact side, and these primordia were able to progress through to later developmental stages (Fig. 4F). We did not observe ground tissue-associated PIN3 expression in the absence of a primordium, suggesting that PIN3 likely acts after FC specification and may promote initiation of LR development on the contact side. This role for PIN3 is consistent with a previous study showing that PIN3 acts in the endodermis to promote LR initiation (30). Analysis of the PIN2 reporter did not reveal obvious differences in expression pattern between contact and air sides of the root (SI Appendix, Fig. S10). These data suggest that PIN transporters could play a role in maintaining local differences in auxin concentration between the air and contact sides but likely do not generate such gradients through differential expression or localization between these sides.

To probe where auxin transcriptional regulation is important for hydropatterning, we used the GAL4/UAS transactivation system to misexpress axr3-1 in different cell layers (31). The axr3-1 mutant allele encodes a dominant suppressor of auxin transcriptional responses (32). We observed significant reductions in hydropatterning in the J0571>>axr3-1 line [cortex (COR) and endodermis (END)], and less in the J0951>>axr3-1 [epidermis (EPI), weak cortex expression] and Q0990>>axr3-1 [stele (STE), no pericycle (PER) expression] lines (Fig. 4G). Thus, ground tissue layers may be important conduits and response centers for auxin during hydropatterning. These data suggest that the perception of local moisture may occur through a cascade of signaling events initiated in the outer tissue layers that are transmitted ultimately to the pericycle.


Historical background

Evidence that prehistoric humans appreciated the form and structure of their contemporary animals has survived in the form of paintings on the walls of caves in France, Spain, and elsewhere. During the early civilizations of China, Egypt, and the Middle East, as humans learned to domesticate certain animals and to cultivate many fruits and grains, they also acquired knowledge about the structures of various plants and animals.

Aristotle was interested in biological form and structure, and his Historia animalium contains excellent descriptions, clearly recognizable in extant species, of the animals of Greece and Asia Minor. He was also interested in developmental morphology and studied the development of chicks before hatching and the breeding methods of sharks and bees. Galen was among the first to dissect animals and to make careful records of his observations of internal structures. His descriptions of the human body, though they remained the unquestioned authority for more than 1,000 years, contained some remarkable errors, for they were based on dissections of pigs and monkeys rather than of humans.

Although it is difficult to pinpoint the emergence of modern morphology as a science, one of the early landmarks was the publication in 1543 of De humani corporis fabrica by Andreas Vesalius, whose careful dissections of human bodies and accurate drawings of his observations revealed many of the inaccuracies in Galen’s earlier descriptions of the human body.

In 1661 an Italian physiologist, Marcello Malpighi, the founder of microscopic anatomy, demonstrated the presence of the small blood vessels called capillaries, which connect arteries and veins. The existence of capillaries had been postulated 30 years earlier by English physician William Harvey, whose classic experiments on the direction of blood flow in arteries and veins indicated that minute connections must exist between them. Between 1668 and 1680, Dutch microscopist Antonie van Leeuwenhoek used the recently invented microscope to describe red blood cells, human sperm cells, bacteria, protozoans, and various other structures.

Cellular components—the nucleus and nucleolus of plant cells and the chromosomes within the nucleus—and the complex sequence of nuclear events (mitosis) that occur during cell division were described by various scientists throughout the 19th century. Organographie der Pflanzen (1898–1901 Organography of Plants, 1900–05), the great work of a German botanist, Karl von Goebel, who was associated with morphology in all its aspects, remains a classic in the field. British surgeon John Hunter and French zoologist Georges Cuvier were early 19th-century pioneers in the study of similar structures in different animals—i.e., comparative morphology. Cuvier in particular was among the first to study the structures of both fossils and living organisms and is credited with founding the science of paleontology. A British biologist, Sir Richard Owen, developed two concepts of basic importance in comparative morphology—homology, which refers to intrinsic structural similarity, and analogy, which refers to superficial functional similarity. Although the concepts antedate the Darwinian view of evolution, the anatomical data on which they were based became, largely as a result of the work of German comparative anatomist Carl Gegenbaur, important evidence in favour of evolutionary change, despite Owen’s steady unwillingness to accept the view of diversification of life from a common origin.

One of the major thrusts in contemporary morphology has been the elucidation of the molecular basis of cellular structure. Techniques such as electron microscopy have revealed the complex details of cell structure, provided a basis for relating structural details to the particular functions of the cell, and shown that certain cellular components occur in a variety of tissues. Studies of the smallest components of cells have clarified the structural basis not only for the contraction of muscle cells but also for the motility of the tail of the sperm cell and the hairlike projections (cilia and flagella) found on protozoans and other cells. Studies involving the structural details of plant cells, although begun somewhat later than those concerned with animal cells, have revealed fascinating facts about such important structures as the chloroplasts, which contain chlorophyll that functions in photosynthesis. Attention has also been focused on the plant tissues composed of cells that retain their power to divide (meristems), particularly at the tips of stems, and their relationship with the new parts to which they give rise. The structural details of bacteria and blue-green algae, which are similar to each other in many respects but markedly different from both higher plants and animals, have been studied in an attempt to determine their origin.

Morphology continues to be of importance in taxonomy because morphological features characteristic of a particular species are used to identify it. As biologists have begun to devote more attention to ecology, the identification of plant and animal species present in an area and perhaps changing in numbers in response to environmental changes has become increasingly significant.


Types of Staining


Simple Staining

It determines the cell shape, size and arrangement of the microorganisms. It is a very quick or simple method to perform and it makes the use of a single stain only. These are of two types, namely direct and indirect staining.

Characteristic Differences Between Direct and Indirect Staining:

CharacteristicsDirect stainingIndirect staining
Stain usedBasic stainAcidic stain
Charge of stainPositiveNegative
ExamplesMethylene blue, crystal violet, carbol fuschinNigrosine, india ink, congo red
OutcomeStains the specimenStains the background
General view after staining
Principle for discolorationBecause of the positively charged stain, it gets attracted towards the negatively charged cell, hence it get fixed to the cell that retain the color of stain results in colorless background with colored cell.Because of the negatively charged stain, it gets repelled by the negatively charged cell, hence it does not fixed to the cell, results in colorless cell with colored background.

Differential Staining

It differentiates between the physical and chemical properties of two different groups of an organism, depending on the cell-wall characteristics. It makes the use of multiple or more than one stains. It can be categorised into two types that are given below:

It provides an important tool to differentiate the two major groups of bacteria, i.e. gram-positive and gram-negative. Dr Hans Christian Joachim Gram introduced this method in 1884. It is carried out by the use of differential stain known as Gram’s stain.

Procedure:

Gram stainingProtocolGram positive bacteriaGram negative bacteria
Primary stainingHeat fixed smear is flooded by crystal violet and allowed to stand for 1min.
MordantingAfter washing, iodine is then flooded and allowed to stand for 1min.
DecolourizationAfter washing, alcohol is added that is washed immediately
Counter stainingAt last, safranin is flooded over the smear and allowed to stand for 30sec, then washed by water.
ObservationAfter air drying, place one drop of oil immersion over the smear and adjust the microscope to identify the specimen, whether it is gram negative or gram positive.
Appear purple in colour because of teichoic acid that resist the primary stain.Appear pink in color due to lack of teichoic acid,alcohol creates pore in the cell which decolourizes the primary stain

It differentiates species of mycobacterium from the other groups of bacteria. Paul Ehrlich first developed it in 1882. And later, this technique was modified by a scientist named Ziehl Neelson.

Procedure

Acid fast stainingProtocolAcid fast bacteriaNon acid fast bacteria
Primary stainingHeat fixed smear is flooded with carbol fuschin and allowed to stand for 1 min.
DecolourizationAfter washing, acid alcohol is added.
Counter stainingAt last, methylene blue is flooded over the smear and allowed to stand for 30 sec, then wash it with water
ObservationAfter air drying, place one drop of oil immersion over the smear and adjust the microscope to identify the specimen, whether specimen is acid fast or not.
Appears red in colour due to presence of mycolic acid that resist the color of primary stain and does not decolourize.Appears blue in colour, as they lack mycolic acid, alcohol creates pore in the cell that decolourizes the primary stain.

Special Staining

It helps in the identification of particular internal and external structural components of the specimen. It includes capsule, endospore and flagella staining.

It differentiates the capsule from the rest of the cell body. This is carried out by the use of both positive and negative dyes.

Capsule: It can define as the polysaccharide envelope, which surrounds the cell wall. Capsule performs many functions like cell protection against desiccation, phagocytic actions and also helps in cell attachment to the host. A capsule is responsible for the pathogenicity or virulence of an organism. It can be seen in the cells of the gram-positive and gram-negative bacteria.

Procedure

Capsule stainingProtocolDiagram
Primary stainingDrop of India ink is placed on a clean slide.
SmearingInoculum is then smeared in a dye.
DraggingUse another slide to drag the mixture into thin film, and then air dried.
Secondary stainingCrystal violet is flooded over the thin film, and then air dried.
ObservationExamine the cells whether they are encapsulated or not.
Interpretation of result
Positive: Zone formation occurs against dark background
Negative: Zone formation does not occur

It differentiates the endospore from the vegetative cell and makes the use of both acidic and basic stains.

Endospore: A term itself defines its meaning, in which endo stands for inside and spore stands for a reproductive structure. Therefore, endospores are the reproductive structures inherent to the cell. It acts like a dormant spore, which can resist harsh physical and chemical conditions. Endospores are commonly found in gram-positive bacteria. According to their position, they are of three types as given below:

Procedure

Endospore stainingProtocolDiagram
Primary stainingMalachite green is flooded over the smear
Heat fixing Then the mixture is heat fixed
DecolourizationDecolourized by water
Counter stainingSafranin is then flooded over the mixture and then air dried
ObservationExamine the slide under the microscope, whether endospore is present or not
Interpretation of result:
Positive: If Endospore present, it will appear green in color whereas vegetative cell appears as pink
Negative: And if endospore is absent then only vegetative cells will appear pink in color

It helps in the identification of the bacterial motility through the presence or absence of flagella. It makes the use of acidic and neutral stain.

Flagella: These are long, thread-like structures, which protrudes outside the cell membrane. Its primary function is to provide motility or locomotion. According to the arrangement, these are of following types:

Atrichous: These are without flagella.
Monotrichous: Single flagellum is present at one end.
Amphitrichous: Single flagellum is present at both the ends.
Lophotrichous: Cluster of flagella are present on one end.
Peritrichous: Flagella are present all over the cell surface.

Procedure

Flagella stainingProtocolDiagram
Primary stainingOne drop of leifson’s stain is flooded over the smear
Secondary stainingAfter that methylene blue is added, and allowed to stand for one minute
ObservationExamine the appearance of flagella to know whether the bacteria is motile or not
Interpretation of result:
Positive: If flagella is present, then it will appear red in color while cell appears blue
Negative: And if not present, only cell will appear blue in color

Examples of Bacteria in different Staining Methods

Applications

  • Staining methods have wide applicability in both biological and biochemical research.
  • It is used in staining of metal.
  • Used in staining of the wood.

Conclusion

Various staining techniques are used for different purposes like to study bacterial morphology and to examine internal and external cellular components. It can also be used to identify the particular group of bacteria, after which we can further classify the type of specimen, based on their growth behaviour and microscopic characteristics.


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