2 Jana Albrechtova: Plant Anatomy in Environmental Physiology, 2004 Jana Albrechtova Plant Anatomy in Environmental Studies . Charles University in Prague Department of Plant Physiology, Faculty of Science 2004 Prague, Czech Republic 3 Jana Albrechtova: Plant Anatomy in Environmental Physiology, 2004 Contents A. Commentary Preface .......................................................................................................................................1 1. Introduction..........................................................................................................................3 1.1. Historical Milestones of Plant Anatomy .........................................................................5 2. Methodological Approaches in Plant Anatomy.................................................................6 2.1. Methods of Plant Histology.............................................................................................6 2.1.1. Conventional staining and histochemical methods ...............................................7 2.1.2. Enzyme histochemical methods............................................................................9 2.1.3. Combination of histochemical methods with other methodological a pproaches .......................................................................................................................................11 2.2. Microscopic imaging..................................................................................................... 12 2.3. Methods of analysis in plant anatomy........................................................................... 13 2.3.1. Descriptive analysis .................................................................................................13 2.3.2. Semi-quantitative analysis .......................................................................................14 2.3.3. Quantitative analysis ...............................................................................................14 2.3.3.1. Historical milestones of quantitative plant analysis.........................................15 2.3.3.2. Stereological methods ......................................................................................16 2.3.3.3. Image analysis..................................................................................................17 2.3.3.4. Review of methodological approaches of quantitative analysis ......................17 2.3.3.4.1. Planar measurements.....................................................................................17 2.3.3.4.2. Estimation of spatial characteristics..............................................................19 2.3.3.5. Image analysis of a colored reaction ................................................................21 2.3.3.6. Design of experiments with quantitative analysis............................................22 3. Concept of scaling in plant biology................................................................................... 23 3.2. Remote sensing in plant anatomy and physiology........................................................ 24 3.3.1. Case study: monitoring Norway spruce health - the NASA project.......................28 4. Conclusions ......................................................................................................................... 29 5. Literature cited................................................................................................................... 30 1 Jana Albrechtova: Plant Anatomy in Environmental Physiology, 2004 Preface We all are ultimately dependent on green plants for our survival on Earth. By converting radiant energy into chemical energy, plants are the basic primary producers in food webs and food chains. As a “waste” product of photosynthesis, plants have been releasing oxygen to the atmosphere for hundred millions of years creating a key life-supporting atmosphere for humans and other aerobic organisms. Last but not least, plants provide a living environment for other organisms. Plant biology usually has received less financial and other support and recognition than the human and animal sciences, which have been always of greater interest since they directly relate to human health. However, progress in plant biotechnologies phytopharmacology, and crop sciences has shown that plant sciences relate to our health, too. During the 20th century it became clear that humans now face global environmental problems caused by their activities, such as global climate change, different types of pollution, loss of bio-diversity, which encompassed plant sciences in a very integral way. At the beginning of the Third Millennium it became clear that investment in plant sciences is crucial for any future scenario of sustainable development for human civilization. According to Passioura (1979), plants are layered, or hierarchically organised, systems. Going down through the levels we might have: ecosystem > community > plant > organ > tissue> cell > organelle > membrane > molecule > sub -molecular. What happens at one level is explained by what happens at the level below and is given significance (or meaning) by what happens at the level above. According to the level of study we can use a different range of methods. Fields of plant biology such as cell biology (or cytology), anatomy, taxonomy, are not sharply distinct from each other, and I agree with Esau (1977) that „in this interrelationship of sciences, anatomy plays a major role“. Studies of structure and function cannot be separated from each other. A realistic interpretation of a plant function being studied can be laid down only on the basis of thorough knowledge of the structure of cells, tissues and/or organs associated with that function. The origins of microscopy and of intensive study in plant anatomy date back many centuries and are described in more detail in the chapter 1. The first monograph about plant anatomy was published by Nehemiah Grew in 1682. Before the boom of new technologies during the second half of the 20th century, plant anatomy was regarded as more or less a rigid, little developing field, which hardly could bring some revolutionary concepts into plant biology. However, as the result of developing new, modern technologies, the whole „new universe“ emerged for the use of plant anatomy, and it is correct to say that a new age of “Rennaissance in Plant Anatomy” arose. Together, quantitative methods and the use of traditional descriptive anatomy give origin to quantitative plant anatomy (rev. by Natr 1988, Pazourek 1988). The introduction of quantitative methods in plant anatomy was stimulated by the needs of other botanical fields, particularly systematic botany, plant physiology and cell biology. During the last half-century the development of physics, mathematics and their technological applications gave rise to informatics and computer sciences. It has allowed the development of new quantitative methods, which later on started to be used in plant anatomy, such as stereological methods and image analysis. Quantitative plant anatomy has a long tradition in the Department of Plant Physiology, Charles University in Prague, as will be shown in the chapter 2.3. The last half-century has been an exciting time for plant biology on the whole and for plant anatomy in particular. Advances in natural sciences and in technologies together with their applications created an increasing demand for experimental plant biology as a means of more accurate and more detailed study of plant structure and function. This new age started to prioritize complex, integral studies enabling the study of plants on different hierarchical levels involving the spectral, physiological, biochemical, structural and molecular approaches and providing a complex insight into the problems under study. Many of the present studies in this thesis have a complex character combining anatomical, histochemical and biochemical methods. Technological developments during the last half-century have brought new imaging concepts, which have completely transformed plant anatomy. First, electron microscopy has 2 Jana Albrechtova: Plant Anatomy in Environmental Physiology, 2004 become a common tool. Then confocal microscopy became a general tool for studying plant anatomical structures enabling, among others, the chance to image thick (about 100mm) biological sections or tiny biological objects in three dimensions. Two-photon or multiphoton excitation microscopy and other nonlinear optical techniques emerged in the 1990’s bringing other exciting possibilities in anatomical research through studying more processes in in vivo and in situ conditions. In the current period of fast development of molecular methods and comparative genome analysis of living organisms, the apparent limit of understanding of their functions is a matter of detailed knowledge of cellular and subcellular structures, dynamic complexity of tissues, and organ structure. We need to study signs of expressions of individual genes at the metabolic and structural levels. For this purpose, the powerful tools from plant anatomical methods became a range of in situ detections and highly sensitive immunofluorescence techniques. All these are dependent on development and application of sensitive methods of image capturing and analysis. The other „extreme“ approaches to the study of plant biology are integral, complex ecological studies, enabling scaling up the information gained on different hierarchical levels, starting with