Cell division and the formation of the new wall The normal asexual method of reproduction is by division of one

cell into two, the valves of the parent cell becoming the epithecas of the daughter cells with each daughter cell producing a new hypotheca (Figs. 17.9, 17.10). As a result of cell division, one of the daughter cells is of the same size as the parent cell, and the other is smaller. As the size of the cell decreases, so does the relative width to height and the morphology of the cell; in other words, the smaller cells are not geometrically proportional to the larger ones from which they arise. Uptake of silica is confined to a period of the cell cycle following cytokinesis and prior to the separation of the two daughter cells (Sullivan, 1977). Cellular energy for silicification and transport comes from aerobic respiration without any direct involvement of photosynthetic energy (Martin-Jezequel et al., 2000). Diatoms have an absolute requirement for silicon if cell division is to take place. In water, solid silica dissociates to produce undissociated silicic acid Si(OH)4: SiO2 (solid) _2 H2O__Si(OH)4. By increasing the concentration in solution with a pH less than 9, or decreasing the pH of a saturated solution, silicic acid will autopolymerize to form amorphous silica. Amorphous silica is the form of silicon in diatom cell walls. Although silicon is the second most abundant element in the Earths crust, its availability is limited by its solubility in water. The growth of marine diatoms can so deplete surface waters of silicon that their further growth is prevented (Hildebrand, 2004). The Si(OH)4 in marine waters is about 6 ppm. the global ocean, about 97% of the dissolved Si is present as Si(OH)4, with the pH of seawater buffered at about 8.0 by the CO2carbonate system (Del Amo and Brzezinski, 1999). However, in freshwater lakes where pH values reach up to 10, Si(OH)4 is only about 23% of the total dissolved silicon, most of the rest being present as ionized silicic acid SiO(OH)3 _ according to the following formula: Si(OH)4_H2O__SiO(OH)3 _ _H_. Silica is taken up into the diatom cell as Si(OH)4 by a protein as an active silicon transporter requiring metabolic energy (Hildebrand et al., 2004; Wetherbee et al., 2004). Silicic acid transport is coupled to sodium in marine diatoms and sodium and perhaps potassium in freshwater diatoms. The transport in marine species has the characteristics of a sodium/silicic acid symporter with a Si(OH)4:Na_ ratio of 1:1. At least five types of silicic acid transporter (SIT) genes have been isolated. Germanium (germanic acid) and silicic acid are competitive inhibitors of each other but germanium can not be incorporated into the diatom cell wall. Prior to cell division, the cell elongates, pushing the epitheca away from the hypotheca, and the nucleus divides. After the protoplasm has divided (Fig. 17.10) into two by the invagination of the plasmalemma, the Golgi bodies produce translucent vesicles which collect beneath the plasmalemma. These vesicles fuse to form the silicalemma or membrane of the silica deposition vesicle (Li et al., 1989). The vesicle gradually expands and assumes the shape of a new valve (Schmid and Volcani, 1983). Two silica deposition vesicles are formed per cell with silica deposited in each to form two new hypovalves (Brzezinski and Conley, 1994). The silicon is probably packaged by Golgi into vesicles that are transported to the silica deposition vesicle by microfilaments in the cytoplasm. At the silica deposition vesicle, the small, silica-laden vesicles fuse with the silicalemma, adding membrane material to the silicalemma, and releasing silica to the interior of the silica deposition vesicle (Fig. 17.10). The silica deposition vesicle determines the ultimate form of the silicified frustule (Fig. 17.15). Silica is deposited as amorphous silica in the form of spheres 3050 nm in diameter (Figs. 17.11, 17.12) (Crawford et al., 2001). The silica deposition vesicles are acidic, the low pH facilitating fast nucleation and aggregation of silica particles (Vrieling et al., 1999). The acidic environment also protects the newly formed valves from dissolution before coverage by an organic casing prior to secretion. The silica deposition vesicles contain peptides called silaffins (Fig. 17.13) that cause the precipitation of silicic acid into silica nanospheres (Figs.17.12, 17.14) (Kroger et al., 1999, 2000; Kroger and Sumper, 2004). Different diatom species have different silaffins that, in turn, have different polyamine chains (Fig. 17.13) attached to the silaffins. Different silaffins produce different sized silica nanospheres and it may be that the specific silaffin controls the frustule ornamentation, a characteristic of the individual diatom species. The discovery of silaffins has generated considerable commercial interest since silica-based materials such as resins, molecular sieves, and catalysts are widely used in industry. The production of the manufactured silica-based materials currently requires extremes of temperature, pressure, and pH. In contrast, biosilicification with silaffins proceeds at ambient temperatures and pressures. A class of glycoproteins, called frustulins, is also associated with the silica deposition vesicle. The frustulins are associated with deposition of the organic casing around the silicified frustules (Perry and Keeling-

Tucker, 2000). When the deposition of silica is complete, the inner membrane of the silicalemma becomes the plasmalemma of the daughter cell, and original plasmalemma and the external membrane of the silicalemma are lost (Fig. 17.10). After the epitheca and the hypotheca are formed, Golgi vesicles now collect and fuse to form the silicalemma of the girdle band. The girdle band is deposited outside the cell in the area between the hypovalve and epivalve when silica deposition is complete. If there are further girdle bands, they are formed in the same manner (Dawson, 1973). Although amorphous silica is slowly soluble at the pH of natural waters, the silica frustule of living diatoms appears to be protected by the layer of organic material that surrounded the frustule. After death, the silica usually dissolves (Bidle and Azam, 1999). In certain environments, however, the walls of planktonic diatoms may settle and accumulate on the bottom faster than they dissolve, thereby forming diatomaceous ooze. Resting spores and resting cells Some diatom cells form thick, ornamented walls at different times in their life cycle and become resting spores (Fig. 17.22) (McQuoid and Hobson,1996). If such cells are planktonic, they fall to the bottom where, presumably, they await more favorable conditions. In Ditylum brightwelli (Gross, 1940), the resting spore is formed by the protoplasm shrinking and the plasmalemma drawing away from the cell wall (Fig. 17.22(a), (b)). The resting spore then formed has a much smaller volume than the original cell, the main decrease being due to the loss of vacuoles and their contents. In germination, the resting spore produces a number of fine protoplasmic strands, which radiate in all directions. Chloroplasts are gradually released from the central mass and move along the processes while the whole protoplast expands. After 2 days, the protoplast has assumed a spherical shape, resembling an auxospore. This protoplast elongates and becomes cylindrical, with a valve appearing at one end within 24 hours and a second valve appearing at the other end after 48 hours. The new cells are much wider than the parent cells. As noted above, the normal method of reproduction, by binary fission, results in a decreased size in the daughter cells. In some species a certain size is reached when sexually or asexually produced auxospores may reestablish the maximum cell size (Nagai et al., 1995). It is, however, known that auxospore formation is generally infrequent, probably taking place once in two or more years in some species. Resting spore formation is therefore a much more frequent method of reestablishment of cell size (Fig. 17.44). Resting spores are formed after the diatom cells have been subjected to a stress shock. In the ocean, light, temperature, and salinity are relatively stable, with sudden changes rarely occurring, even though there are seasonal changes. In the marine environment, it is usually stress shocks from nutrient depletion that trigger resting spore formation (Davis et al., 1980). Because of the exponential nature of phytoplankton growth, the limiting nutrient is reduced from a considerable level to near zero during the last doubling of the phytoplankton at the end of a bloom (in 1 day or less for many diatoms). Thus a sudden nutrient shock is a typical feature at the end of a diatom bloom. Marine centric diatoms commonly form morphologically different resting spores, whereas there are only a few pennate diatoms that do. Eunotia soleirolii is one of the few freshwater pinnate diatoms that form resting spores (von Stosch and Fecher, 1979). In the freshwater environment, diatoms are subjected to more environmental shocks than in the marine environment. Eunotia soleirolii will form resting spores when subjected to (1) high temperatures (24 C); (2) high or low pH; or (3) iron, silica, phosphate, or nitrate deficiencies. In many other diatoms, resting spores germinate when placed in fresh medium. However, the resting spores of E. soleirolii require dormancy to be broken by a dark period at 2 C to 15 C for a minimum of 4 to 5 weeks. Thus E. soleirolii thrives in colder seasons in northern Europe and is induced to form resting spores by rising temperatures and perhaps concomitant nutrient deficiencies. The resting spores are viable for up to 3 years, but would normally last the summer before dormancy would be broken by the cold temperature of autumn, resulting in germination of the resting spores. Resting cells have the same morphology as vegetative cells and do not form a protective layer, thereby differing from resting spores. Growing vegetative cells of Amphora coffaeformis require 4 weeks in the dark to form resting cells (Anderson, 1975). Formation of resting cells initially involves autophagic activity, with the breakdown of existing structures. Large vacuoles decrease in size, and many small ones develop; the mitochondria become fewer; and large lipid bodies are formed (Fig. 17.22). The resting cells contain as much chlorophyll as the vegetative cells, and the whole cell appears to be a parsimonious assemblage of organelles prepared to resume metabolism and growth upon return to favorable conditions. During the summer, diatom cells sink into deep water below the euphotic zone, where they form resting cells and become

dormant. Such cells remain viable for at least 2 months in such an environment (Anderson, 1976). Their respiratory rate is 20% that of normal cells, and their photosynthetic capability is very low. Vertical mixing brings these cells and nutrient-rich water to the surface, and within 2 days the cells become active and begin to reproduce. Viable resting cells of diatoms have been collected at 6150-m water depth in the North Atlantic. Beta Carotene Beta-carotene belongs to the group of pigments called carotenoids. Carotenoids are a class of natural fat-soluble pigments found principally in plants, algae, and photosynthetic bacteria, where they play a critical role in the photosynthetic process. In human beings, carotenoids can serve several important functions. The most widely studied and well-understood nutritional role for carotenoids is their provitamin-A activity. Beta-carotene is converted to Vitamin A in the body. Until 1980, production of beta-carotene was synthetic. Natural carotenoids, although more expensive than synthetic have the advantage of supplying the natural isomers in their natural ratio and the natural isomers of beta-carotene are considered superior to the trans-synthetic form. In 1994 algal beta-carotene was sold in small quantities. Sources of Beta-Carotene Good sources of beta-carotene include dark green and orange-yellow vegetables, such as carrots, sweet potatoes, squash, spinach, broccoli, romaine lettuce, apricots, and green peppers. Dunaliella salina, a marine microalga is a rich source of beta-carotene, alpha-carotene, cryptoxanthin, zeaxanthin, lutein and lycopene. The average concentration of carotenoids in most algae is only 0.12%, but Dunaliella when grown under the right conditions of high salinity and light intensity will produce up to 14% beta-carotene. Dunaliella is, therefore, well suited to the commercial production of beta-carotene and several industrial production plants are in operation around the world including Australia, Israel, USA, India and China. Algae Strains for Beta-Carotene Production Dunaliella (D salina, D bardawil, D kona) Spirulina platensis Chlorella Caulerpa taxifolia Commercial Production of Beta-carotene Dunaliella has the ability to grow at very high salt concentrations where few other organisms can survive. Its high temperature tolerance (up to about 40C), and the high cell content of beta-carotene (up to 14% of dry wt.) made this alga an attractive candidate for commercial production of betacarotene. The extreme conditions under which this alga grows means that relatively simple cultivation systems can be used. Dunaliella production facilities are located in areas where solar irradiance is maximal, cloudiness is minimal, climate is warm, and hypersaline water available. Dunaliella for beta-carotene production has been cultivated in large shallow ponds. These ponds have a depth of between 30 and 60cm and are only mixed by wind and thermal convection. The harvested biomass is extracted and pure beta-carotene or mixed carotenoids are sold as a nutritional supplement and natural food coloring. Dried Dunaliella powder is also sold as a feed additive for aquaculture to pigment crustaceans such as prawns.

Fuels The lipid, or oily part of the algae biomass can then be extracted and converted into biodiesel through a process similar to that used for any other vegetable oil, or converted in a refinery into "drop-in"

replacements for petroleum-based fuels. The algae's carbohydrate content can be fermented into bioethanol and biobutanol.[17] Biodiesel The U.S. Department of Energy's Aquatic Species Program, 19781996, focused on biodiesel from microalgae. The final report suggested that biodiesel could be the only viable method by which to produce enough fuel to replace current world diesel usage.[18] If algae-derived biodiesel were to replace the annual global production of 1.1bn tons of conventional diesel then a land mass of 57.3 million hectares would be required, which would be highly favorable compared to other biofuels. [19] As they do not have to produce structural compounds such as cellulose for leaves, stems, or roots, and because they can be grown floating in a rich nutritional medium, microalgae can have faster growth rates than terrestrial crops. Also, they can convert a much higher fraction of their biomass to oil than conventional crops, e.g. 60% versus 2-3% for soybeans.[20] The per unit area yield of oil from algae is estimated to be from between 4,700 to 18,000 m3/km2/year (1,000 to 6,500 US gallons/acre/year).[21] This is 7 to 30 times greater than the next best crop, Chinese tallow (650 m3/km2/year, or 700 US gal/acre/year).[dubious discuss] Studies[20] show that some species of algae can produce up to 60% of their dry weight in the form of oil. Because the cells grow in aqueous suspension, where they have more efficient access to water, CO2 and dissolved nutrients, microalgae are capable of producing large amounts of biomass and usable oil in either high rate algal ponds or photobioreactors. This oil can then be turned into biodiesel which could be sold for use in automobiles. Regional production of microalgae and processing into biofuels will provide economic benefits to rural communities.[22] Biobutanol : Butanol can be made from algae or diatoms using only a solar powered biorefinery. This fuel has an energy density 10% less than gasoline, and greater than that of either ethanolor methanol. In most gasoline engines, butanol can be used in place of gasoline with no modifications. In several tests, butanol consumption is similar to that of gasoline, and when blended with gasoline, provides better performance and corrosion resistance than that of ethanol or E85.[23] The green waste left over from the algae oil extraction can be used to produce butanol. In addition, it has been shown that macroalgae (seaweeds) can be fermented by Clostridria to butanol and other solvents.[24] Biogasoline : Biogasoline is produced from biomass such as algae. Like traditionally produced gasoline, it contains between 6 (hexane) and 12 (dodecane) carbon atoms per molecule and can be used in internal-combustion engines. Methane : Methane,[25] the main constituent of natural gas can be produced from algae in various methods, namely Gasification, Pyrolysis and Anaerobic Digestion. In Gasification and Pyrolysis methods methane is extracted under high temperature and pressure. Anaerobic Digestion[26] is a straight forward method involved in decomposition of algae into simple components then transforming it into fatty acids using microbes like acidific bacteria followed by removing any solid particles and finally adding methanogenic bacteria to release a gas mixture containing methane. A number of studies have successfully shown that biomass from microalgae can be converted into biogas via anaerobic digestion.[27][28][29][30][31]Therefore, in order to improve the overall energy balance of microalgae cultivation operations, it has been proposed to recover the energy contained in waste biomass via anaerobic digestion to methane for generating electricity.[32]

Ethanol : The Algenol system which is being commercialized by BioFields in Puerto Libertad, Sonora, Mexico utilizes seawater and industrial exhaust to produce ethanol. Vegetable oil fuel : Algal-oils could potentially be used as vegetable oil fuel. Hydrocracking to traditional transport fuels : Algae can be used to produce 'green diesel' (also known as renewable diesel, hydro-treated vegetable oil[33] or hydrogen-derived renewable diesel)[34] through a hydrocracking refinery process that breaks molecules down into shorter hydrocarbon chains used in diesel engines.[33][35] It has the same chemical properties as petroleum-based diesel[33]meaning that it does not require new engines, pipelines or infrastructure to distribute and use. It has yet to be produced at a cost that is competitive with petroleum.[34] Jet fuel : Rising jet fuel prices are putting severe pressure on airline companies, [36] creating an incentive for algal jet fuel research. The International Air Transport Association, for example, supports research, development and deployment of algal fuels. IATA's goal is for its members to be using 10% alternative fuels by 2017.[37] Trials have been carried with aviation biofuel by Air New Zealand,[38] Lufthansa, and Virgin Airlines.[39] In February 2010, the Defense Advanced Research Projects Agency announced that the U.S. military was about to begin large-scale oil production from algal ponds into jet fuel. After extraction at a cost of $2 per gallon, the oil will be refined at less than $3 a gallon. A larger-scale refining operation, producing 50 million gallons a year, is expected to go into production in 2013, with the possibility of lower per gallon costs so that algae-based fuel would be competitive with fossil fuels. The projects, run by the companies SAIC andGeneral Atomics, are expected to produce 1,000 gallons of oil per acre per year from algal ponds.

Single Cell Protein (SCP) and Mycoprotein Production of Algal Biomass Algae (cyanobacteria and unicellular eukaryotes) grow autotrophically and synthesize their food by taking energy from sunlight or artificial light, carbon source from carbon dioxide, and nutrients from carbohydrates present in growth medium. In a few countries, cultivation of algae is carried -out in large trenches i.e. particularly in sewage oxidation ponds by using sunlight (Oswald, 1969) or in an artificial illumination conditions for use in life supportive systems for extended space exploration (Litchfield, 1967). Chlorella strains are being used for a variety of applications in biotechnology. Due to their very high protein contents, they serve to improve protein deficiency and can be used as feed for production of animal protein. In many countries strains of Chlorellaare utilized for sewage oxidation and waste water treatment (Kessler, 1989). For cultivation of algae on sewage wastes, oxidation ponds are prepared, where sewage is allowed to accumulate. It is awaited till mixed cultures of algae grow (or inoculated with a singly prepared algal culture). For example, in Japan mixed culture ofChlorella ellipsoides and Scenedesmus obliguus was developed in open pond

systems. Factors Affecting Biomass Production Following are the factors affecting the yield of biomass : (i) Illumination time; (ii) Light intensity; (iii) Supply of CO2. Concentrations of CO2 differ in different conditions, for example, an alkaline lake. Lake Texcoco in Mexico, has high concentration of sodium carbonate. On the other hand, algal growth is limited as a result of liberation of CO 2and ammonia by bacterial activity; (iv) Nitrogen sources (ammonium salts or nitrates are the suitable nitrogen sources which increase biomass yield); (v) Agitation of growing cells to maintain cells in suspension. Biomass yield ranges between 12 and 15 g/m2/day (on dry weight basis) as obtained with Spirulina maxima and Scenedesmus quadricauda grown in out door pond conditions (Clement, 1968). Mass cultivation of algae has been started in many countries, such as Japan, West Germany (now Germany), Mexico, Chechoslovakia, India, etc. In India, National Environmental Engineering Research Institute (NEERI), Nagpur has developed a technique of cultivating algae in sewage oxidation pond systems. This practice is also in use at NBRI (Lucknow), Hyderabad and other centres. Interestingly, experiments conducted at the CFTRI, Mysore have shown that the microalgae e.g. Scenedesmus acutus and Spirulina platensis could be cultivated on a large scale and used as food and feed as they are rich in protein and their nutrient value is comparable to conventional foods. A flow diagram of use of different groups of algae at various stages in waste water ponds and possible application of algal biomass is shown in Fig. 18.1. Advantages of Producing Microbial Protein Roth (1982) has described a number of advantages in the production of microbial protein, compared to protein problems of conventional crops used as food and feed. These include : (i) Rapid succession of generations (algae, 2-6h; yeast, l-3h; bacteria, 0.5-2h); (ii) Easily modifiable genetically (e.g. for composition of amino acids); (iii) High protein content of 43-85 per cent in the dry mass; (iv) Broad spectrum of original raw material used for the production, which also includes waste products; (v) Production in continuous cultures, consistent quality not dependent on climate in determinable amount, low land requirements, ecologically beneficial. Other advantages are : (a) high solar energy conversionefficiency per unit area (net production in cultivated land-290 gC/m2/day, lakes and streams - 225 gC/m2/day ; estuaries 810 gC/m2/day), (b) easy regulation of environmental factors e.g. physical, nutritional, etc. which maximize solar energy conversion efficiency and yield (c) cellular, molecular and genetic alterations and (d) algal culture in space, which is normally unused instead of competing for land (Vijayan, 1988).

Nutritional Value of SCP Nowadays, considerable information is available on the composition of microbial cells e.g. protein, amino acid, vitamin, and minerals (Litchfield, 1979). Commercial value of SCP depends on their nutritional performance and nevertheless, it has to be evaluated to the prevalent feed protein. SCPs either from alkanes or methanols, are characterized by good content and balance in essential amino acids (Senez, 1986). Composition of growth medium governs the protein and lipid contents of microorganisms. Yeasts, moulds and higher fungi have higher cellular lipid content and lower nitrogen and protein contents, when grown in media having high amount of available carbon as energy source and low nitrogen (Litchfield, 1979). Ignoring a few extreme values, the mean crude protein in dry matter of algae and yeasts, on conventional substrates, lies between 50 and 60 per cent, for alkane yeasts between 55 and 65 per cent, and for bacteria about 80 per cent. A high content of nucleic acid free protein is extremely important for the economic efficiency of the procedure in SCP production. Because of high protein and fat contents, the contribution of carbohydrates to the nutritional value of SCP is not of prime importance. The crude ash content is determined in particular by the nutrient salts of the fermentation medium (Roth, 1982). Estimation of crude protein is based on total nitrogen which is multiplied by the factor 6.25. The protein content of microorganisms computed in this manner does not give the exact figure of protein content, as in the estimation of total nitrogen, the value of nucleic acid is also included which is somewhat erroneous. The most important measure of nutritional value is the actual performance of SCP products as determined in feeding studies. The determinants of the utility of SCP product for application as food for human beings and feed for animals differ. For human beings, protein digestibility and protein efficiency ratio (PER), biological value or net protein utilization (NPU), determined in rats, are the parameters for food application, whereas for animals, metabolizable energy, protein digestibility and feed conversion ratio (weight of ration consumer/weight gain) are the measures or performance in broiler, chickens, swine and calves (and egg laying in hens). Digestibility (D) is the percentage of total nitrogen consumed, which is absorbed through the alimentary tract. It is calculated as below: Ni D Fn = Ni where, Ni = nitrogen ingested from SCP. Fn = nitrogen content in faeces after feeding SCP ULTRASTRUCTURE OF PLASTIDS The basic type of plastid in the algae is a chloroplast, a plastid capable of photo

synthesis. Chromoplast is syn onymous with chloro plast; in the older literature a chloro plast that has a color other than green is often called a chromo plast. A proplastid is a reduced plastid with few if any thylak oids. A pro plastid will usually develop into a chloroplast although in some hetero trophic algae it remains a pro plastid. A leucoplast or amyloplast is a colorless plastid that has become adapted for the accumula tion of storage product. In the Rhodophyta and Chlorophyta, the chloro plasts are bounded by the double membrane of the chloro plast envelope (Fig. 1.12(a), (e)). In the other eukaryotic algae, the chloro plast envelope is surrounded by one of two membranes of chloroplast endo plasmic reticulum (chloroplast E.R.), which has ribosomes attached to the outer face of the membrane adjacent to the cytoplasm. The chloroplast E.R. is the remnant of the food vacuole membrane and/or the plasma membrane involved in the original endosymbiosis leading to the chloroplasts in a secondary endosymbiosis. In the Euglenophyta and Dinophyta, there is one membrane of chloroplast E.R. (Fig. 1.12(c). In the Cryptophyta, Prymnesiophyta, and Heterokontophyta, there are two membranes of chloroplast E.R., with the outer membrane of chloroplast E.R. usually continuous with the outer membrane of the nuclear envelope, especially if the chloroplast number is low (Fig. 1.12 (b), (d)). The basic structure of the photosynthetic apparatus in a plastid consists of a series of flattened membranous vesicles called thylakoids or discs, and a surrounding matrix or stroma. The thylak oids contain the chlorophylls and are the sites of the photochemical reactions; carbon dioxide fixation occurs in the stroma. The thylak oids can be free from one another or grouped to form thylakoid bands. In the cyanobacteria and Rhodophyta (Fig. 1.12(a)), the thylak oids are usually free from one another, with phycobilisomes (containing the phycobiliproteins) on the surface of the thylak oids. The phycobilisomes on the surface of one thylak oid alternate with those on the surface of an adjacent thylak oid. The phycobilisomes appear as 35-nm granules when phycoerythin predominates, or as discs when phycocyanin predominates. In the more primitive members of the Rhodophyta the thylak oids terminate close to the chloroplast envelope, whereas in advanced members of the Rhodophyta peripheral thylak oids are present, which enclose the rest of the thylak oids. In the Cryptophyta, the chloro - plasts contain bands of two thylak oids (Fig. 1.12(b)); the phycobiliproteins are dis persed within the thylak oids. In the Euglenophyta and Heterokontophyta the thylak oids are grouped in bands of three with a girdle or peripheral and running parallel to the Chloroplast envelope. In the Dinophyta, Prymnesiophyta, and Eustigmatophyceae, the thylak oids are also in bands of three, but there is no girdle band (Fig. 1.12(c), (d)). In the Chlorophyta, the thylak oids occur in bands of two to six, with thylak oids running from one band to the next. The above grouping of algal thylak oids into bands occurs under normal growth condi tions. Abnormal growth conditions commonly cause lumping of thylakoids and other variations in structure. A pyrenoid (Fig. 1.12(b)) is a differentiated region within the chloroplast that is denser than the surrounding stroma and may or may not be traversed by thylakoids. A pyrenoid is frequently associated with storage product. Pyrenoids contain ribulose-1, 5-bisphosphate Carboxylase/oxygenase (Rubisco), the enzyme that fixes carbon dioxide (Jenks and Gibbs, 2000; Nagasato et al., 2003). Consequently, the size of the pyrenoid will vary depending on how much Rubisco is present. Rubisco exists in two forms (Jenks and Gibbs, 2000; Zhang and Lin, 2003):

1. Form I occurs in some bacteria, the cyanobacteria, in all green plants and nongreen plants. Form I is composed of eight large subunits and eight small subunits (Fig. 1.13). Form I has a high affinity for CO2 and a low catalytic efficiency (low rate of CO2 fixation). In green algae, euglenoids, and green plants,the large subunit is coded by chloroplast DNA and the small subunit by nuclear DNA. In the cyanelle (endosymbiotic cyanobacterium) of Cyanophora paradoxa and In some non-green algae, both subunits are coded by chloroplast DNA. 2. Form II occurs in some eubacteria and in the dinoflagellates and is composed of two large sub units. Form II has a low affinity for CO2 and a high catalytic efficiency. The common ancestor of all ribulose-1,5-bisphosphate carboxylase was probably similar to Form II and was adapted to the anaerobic condi tions and high CO2 concentra tions pre vailing in the ancient earth (Haygood, 1996). Form I evolved as the earths atmosphere became oxy gen ated, and CO2 concentra tion declined and with it the need for a greater affinity for CO2. The greater affinity for CO2 in Form I, however, came at the price of reduced catalytic efficiency. Chloroplasts contain small (30100 nm), spherical lipid droplets between the thylakoids (Fig. 1.12 (c), (d)). These lipid droplets serve as a pool of lipid reserve within the chloroplast. Many motile algae have groups of tightly packed carotenoid lipid-globules that constitute an orange-red eyespot or stigma (Fig. 5.2) that is involved in response to light. In the Chlorophyta (Fig. 5.2), Cryptophyta (Fig. 9.4) and most of the Heterokontophyta (Fig. 10.1), the eyespot occurs as lipid droplets in the chloroplast. In the Euglenophyta (Fig. 6.2), Eustigmato - phyceae (Fig. 12.1), and Dinophyta (Figs. 7.21, 7.22, 7.23), the eyespot occurs as a group of membrane-bounded lipid droplets, free of the chloroplast. Most chloroplasts contain prokaryotic DNA in an area of the chloroplast devoid of 70S ribosomes (Figs. 1.16 and 1.17). The DNA is an evolu - tionary remnant of the cyanobacterium involved in the endosymbiosis leading to the chloroplast. The individual DNA microfibrils are circular, are attached to the chloro plast membranes, and lack basic pro teins (histones). The algae can be divided into two general groups according to the distri - bution of DNA in the plastids (Coleman, 1985). In the first group, the clumps of DNA (nucleoids) are scattered through out the plastids. This group includes the Cryptophyta, Dinophyta, Prymnesio - phyta, Eustigmatophyceae, Rhodophyta, and Chlorophyta. In the second group, the DNA occurs in a ring just within the girdle lamella. This group includes the Chrysophyceae, Bacillario phyceae, Raphidophyceae, and Xanthophyceae (with the exception of Vaucheria and three genera known to lack girdle lamellae Bumilleria, Bumilleriopsis, and Pseudobumilleriopsis). The Euglenophyta fit into neither group, showing a variable distribution of chloro plast DNA. The photo syn thetic algae have chloro phyll in their chloro plasts. Chlorophyll is composed of a porphyrinring system that is very similar to that of hemo globlin but has a magnesium atom instead of an iron atom (Fig. 1.18). The algae have four types of chloro phyll, a, b, c (c1 and c2), and d. Chlorophyll a is the primary photo syn thetic pigment (the light receptor in photo system I of the light reac tion) in all photo syn thetic algae and ranges from 0.3% to 3.0% of the dry weight. Chlorophyll a is insoluble in water and petroleum ether but soluble in alcohol,

diethyl ether, benzene, and acetone. The pigment has two main absorp tion bands in vitro, one band in the red light region at 663 nm and the other at 430 nm (Fig. 1.19). Whereas chloro phyll a is found in all photo - syn thetic algae, the other algal chloro phylls have a more limited distribution and func tion as accessory photosynthetic pigments. Chlorophyll b is found in the Euglenophyta and Chlorophyta (Fig. 1.18). Chlorophyll b functions photosynthetically as a light-harvesting pigment trans ferring absorbed light energy to chloro phyll a. The ratio of chloro phyll a to chloro phyll b varies from 2:1 to 3:1. The solubil ity character istics of chloro phyll a are similar to chloro phyll b, and in vitro chloro - phyll b has two main absorp tion maxima in acetone or methanol, one at 645 nm and the other at 435 nm (Fig. 1.19). Chlorophyll c (Fig. 1.18) is found in the Dinophyta, Cryptophyta, and most of the Heterokontophyta. Chlorophyll c has two spectrally different components: chloro phyll c1 and c2. chlorophyll c2 is always present, but chloro phyll c1 is absent in the Dinophyta and Cryptophyta. The ratio of chloro phyll a to chloro phyll c ranges from 1.2:2 to 5.5:1. Chlorophyll c probably func tions as an accessory pigment to photo system II. The pigment is soluble in ether, acetone, methanol, and ethyl acetate, but is insoluble in water and petroleum ether. Extracted chloro phyll c1 has main absorp tion maxima at 634, 583, and 440 nm in methanol, whereas chloro phyll c2 has maxima at 635, 586, and 452 nm. Chlorophyll d (Fig. 1.18) occurs in some cyanobacteria Murakami et al., 2004). It has three main absorption bands at 696, 456, and 400 nm. The photosynthetically active pigments of algae are gathered in dis crete pigmentprotein complexes which can be divided func tion ally into two groups (Grossman et al., 1990): 1 the photochemical reac tion center containing chloro phyll a, where light energy is converted into chem ical energy; 2 the light-harvesting complexes that serve as antennae to collect and trans fer avail able light energy to the reac tion center. The light-harvesting complexes use different antennae pigment complexes to capture light energy. All of the light-harvesting complexes are composed of three-membrane spanning helices (Fig. 1.20). 1 Green algae and higher plants use chlorophyll a/b binding proteins. 2 Brown and golden-brown algae, (diatoms, chrysophytes, dinoflagellates, brown algae, and related groups) use a fucoxanthin chloro phyll a/c complex that is an integral part of the thylak oid membrane. The ratio of fucoxanthin to chloro phyll in this complex is approximately 2 : 1 and the character istic brown or goldenbrown color of these algae is due to the high level of fucoxanthin in these cells. Due to chloro phyll c and special xanthophylls, these organ isms are especially suited to harvest blue and green light, which are the most abundant at increasing ocean depths. This lightharvesting complex also is composed of three membrane-spanning helices and is closely related to the lightharvesting complex in the first group (Caron et al., 1996). 3 Cyanobacteria, cryptophytes and red algae use the phycobi li some as the major lightharvesting complex. Carotenoids are yellow, orange, or red pigments that usually occur inside the plastid but may be outside in certain cases. In general, naturally occurring carotenoids can be divided into two classes: (1) oxygen-free hydrocarbons, the carotenes; and (2) their oxygenated derivatives, the xanthophylls. The most widespread carotene in the algae is _-carotene (Fig. 1.21). There are a large number of different xanthophylls, with the Chlorophyta having xanthophylls that most

closely resemble those in higher plants. Fucoxanthin (Fig. 1.21) is the principal xanthophyll in the golden-brown algae (Chrysophyceae, Bacillariophyceae, Prymne - sio phyceae, and Phaeo phyceae), giving these algae their characteristic color. Like the chlorophylls, the carotenoids are soluble in alcohols, benzene, and acetone but insoluble in water. The cyanobacteria and chloroplasts of the hodophyta and Cryptophyta have evolved membrane- peripheral antenna complexes containing phycobiliproteins that transfer light energy to photosytem II reaction centers. Like chlorophyll b/c/d, the phycobiliproteins expand the range of light energy that can be utilized in photosyn - thesis. Light tends to become blue-green as it courses down the water column, and this light is better absorbed by the biliproteins than chlorophyll a.

Pollen

vs

Spore

Diploid spore mother cells give rise to spores. Spores are haploid structures. They are important for reproduction as well as for survival in the unfavorable conditions. Spores are seen as a part of the life cycle of many organisms including plants, fungi, bacteria, algae etc. In plants, depending on the different types of spores, a plant can be homosporous or heterosporous. If the plant has only one type of spores, it is known as homospory. If the plant has two types of spores that are male and female spores, it is known as heterospory. Spore Almost all seed bearing plants are heterosporous. They possess large spores, which are called megaspores in the megasporangium, and small spores, which are called microspores in the microsporangium. As the spores grow they become gametophytes. The megaspores become female gametophytes and the microspores become the male gametophytes. Unlike in primitive plants, in seed bearing plants, the gametophytes are never released from the spore. This can be considered as an evolutionary advance. Due to this nature the gametophytes are well protected from desiccation. But the male sperms produced from the male gametophyte needs to reach the female egg. This is done through the dispersal of spores. Spores can be dispersed by wind, water, or insects. Pollen Male spores are called microspores. Microspores are also called pollen grains. In flowering plants, microspores are found inside the pollen sac or the microsporangium. Microspores are very small, minute structures. They are almost like dust particles. Each microspore has one cell and two coats. Outermost coat is the extine, and the inner one is the intine. Extine is a tough, cutinized layer. Often it contains spinous outgrowths. Sometimes it can be smooth, as well. The intine is smooth, and it is very thin. It is mainly made up of cellulose. The extine contains one or more thin places known as the germ pores through which the intine grows out to form the pollen tube. The pollen tube elongates trough the gynoecium tissues carrying two male gametes in it. Pollen tube grows down and enters the ovule through the micropyle. Then the apex of the pollen tube degrades and the two male nuclei are released in to the ovule. Double fertilization takes place by the fusion of the one male nucleus with the egg cell nucleus, giving rise to the diploid zygote, and fusion of the other male nucleus with the diploid secondary nucleus giving rise to the triploid primary endosperm nucleus. What is the difference between Spores and Pollens? Spores are reproductive haploid structures and which can be large female spores, which are called megaspores, or small male spores, which are called microspores (pollens). In other words, all pollens are spores, but not all spores are pollens. Pollens are produced from the microspore mother cells, but female spores are

produced by the megaspore mother cells. Pollen grains have two outer coats extine and intine and female spores do not have the extine or intine. Pollens are dispersed by various mechanisms, but female spores are retained within the ovary. Pollens are found inside the pollen sac, and female spores are found inside the ovule.

PLANT PATHOLOGY

Phytoalexins. Phytoalexins have been known for several decades to be produced by plants under attack but few fungal enzymes have been found that degrade them during fungal attack. One such enzyme is pisatin demethylase, which is produced by the fungus Nectria haematococca and degrades the pea phytoalexin pisatin. Pisatin demethylase is encoded by one of six such genes of the fungus but disruption of the gene caused only a slight reduction in pathogenicity. However, disruption of one out of four fungal genes that detoxify the phytoalexin maakiain from chickpea resulted in a reduction of pathogenicity, whereas the insertion of additional copies of the same gene in the pathogen isolates resulted in greater disease severity. Some fungal genes protect the fungus and its pathogenicity even after it is growing inside the plant. Numerous such genes are involved in the efflux and influx of fungal molecules into the plant. Disruption of such a gene in M. grisea resulted in loss of pathogenicity. Because the same gene is induced by toxic drugs and by the rice phytoalexin sakuranetin, perhaps it plays a role in the efflux of plant metabolites from the fungus. Because some fungal pathogenicity genes, when mutated, result in auxotrophic strains, it is apparent that levels of nutrients can affect the ability of fungi to colonize plants. It has been known for many years that auxotrophy is linked to a lack of pathogenicity in the corn smut fungus Ustilago maydis, whereas adenine auxotrophs of the apple scab fungus Venturia inaequalis are nonpathogenic on apple. Similarly, auxotrophs of Fusarium sp. in arginine and of Stagonospora sp. in ornithine decarboxylase also lost their ability to cause disease. Introduction Higher plants are routinely exposed to microorganisms, both above and below the ground. Fortunately, only a handful of them cause diseases on them. Indeed, as with animals, compatibility of micro-organisms (susceptible) with the host is an exception in the nature, while incompatibility (resistance) is the rule (Panopoulus et al., 1984). In many cases, following an attack, an encounter leaves no obvious trace of its occurrence and the microbe fails to establish itself due to a lack in activation of pathogenicity functions or to highly effective plant defence mechanisms. Others leave evidence of an intense host-pathogen interaction that eventually results in the restriction of the pathogen (Delaney,2 Figen Mert-Trk 1997). In this case, host tissues often display activated defence functions that produce antimicrobial compounds, enzymes and structural reinforcement that may limit pathogen growth (Dixon and Lamb, 1999). Activated (infection-induced) defence mechanisms operates only once the pathogen attempts to infect the host, thus their products are normally either absent from healthy plant tissues, or present only in lower amounts than that can be detected during incompatible interactions. They therefore provide an excellent model for investigation of events involved in cellular signalling, and to evaluate the role played by specific defence mechanisms in resistance (Smith, 1996). Phytoalexin concept Antimicrobial compounds from plants are broadly classified into two categories: phytoantipicins and phytoalexins (Mansfield, 1999). Phytoanticipins are described as "low molecular weight, antimicrobial

compounds that are present in plants before challenge by micro-organisms, or are produced after infection solely, from pre-existing precursors". Phytoalexins are defined as "low molecular weight, anti-microbial compounds that are both synthesized and accumulated in plants after exposure to microorganisms or abiotic agents" (Paxton, 1980; VanEtten et al., 1994). Phytoalexins represents one component of a battery of induced defence mechanisms used by plants including lytic enzymes such as chitinases and glucanases, oxidizing agents, cell wall lignifications and a number of pathogenesis-related (PR) proteins and transcripts of unknown functions (Dixon and Lamb, 1990; Lamb et al., 1989). It is important to recognise that phytoalexin accumulation may be part of a co-ordinated defence strategy, in which any one factor may alone be unable to account for restriction of the potential pathogen (Mansfield, 1999). Elicitors of phytoalexin accumulation The molecules that signal plants to begin the process of phytoalexin synthesis are called elicitors. Elicitors of biotic origin may be involved in the interaction of plants and potential pathogens, whereas abiotic elicitors are not involved in normal host-pathogen interactions (Darvill and Albersheim, 1984). In natural conditions, the stimulus is provided by the presence of the micro-organism and its perception by the host initiates the chain of events leading to phytoalexin synthesis. Biotic elicitors may originate in the invading organism, in which case they are referred to as "exogenous", whereas "endogenous" elicitors are of plant origin and are generated by the interaction between micro-organism and plant. Molecules with elicitor activity have been identified across a wide range of structural types including polysaccharides, glycoproteins, lipids, lipopolysaccharides, oligosaccharides and even enzymes, though their activity can be attributed to their effect in releasing elicitorctive components from the cell walls of the pathogen or host (Anderson, 1989; Blein et al., 1991; Hahn et al., 992; Ricker and Bostock, 1992; Alghisi and Favaron, 1995). Abiotic elicitors form a diverse collection of molecules that are not derived from natural sources, such as the tissues of the pathogen or host. Under normal circumstances, they would not be encountered by the plant. The group includes compounds such as fungicides; salts of heavy metals, for example Cu2+ and Hg2+; the detergents, basic molecules such as polylysine and histone; reagents that are intercalated DNA (Dixon et al., 1983; Darvill and Albersheim, 1984). Treatment of plant tissues with factors that cause stress, for example repeated freezing and thawing, wounding or exposure to UV light (Kodama et al., 1988; Kodama et al., 1992; Mert-Trk et al., 1998) can also induce phytoalexin synthesis. Phytoalexins in disease resistance Phytoalexins accumulate at infection sites and they inhibit the growth of fungi and bacteria in vitro therefore, it is logical to consider them as possible plant-defence compounds against diseases caused by fungi and bacteria. Depending upon the phytoalexin, fungus and bioassay, the EC50 for fungi is generally 10-3 to 10-5 M (reviewed by Kuc, 1995). Thus they are comparatively weak as antifungal agents. Although there is no evidence that are phytoalexins are translocated, the speed of their accumulation andPhytoalexins in plant stress 3 localization at the infection site may permit the pathogen to encounter concentrations far in excess of the EC50 at early stages in the infection process. There is evidence for this presumption. Concerning the accumulation of pisatin in pea and phaseollin in green bean, it was apparent that the phytoalexins accumulated to fungitoxic concentrations not only in inoculum droplets placed on opened pea or bean pods but also in the tissues immediately below the inoculum droplets (Cruickshank and Perrin, 1968). These data supported a role for phytoalexins in plant disease resistance, but there were and still are exceptions. There are also examples that phytoalexins accumulated during compatible plant-pathogen interactions. These include the induction of pisatin by the virulent Oomycete Aphanomyces eutiches (Pueppke and VanEtten, 1976) and by the pathogenic strains of the fungus Nectria hematococca and induction of spirobrassinin by virulent races of Leptosphaeria maculans (Pedras and Seguin-Swartz, 1992). Similarly Glazebrook and Ausubel (1994) reported that the virulent pathogen Pseudomonas syringae pv. maculicola elicits the synthesis of high levels of camalexin in Arabidopsis thaliana. Mert-Trk et al. (1998) also showed that camalexin accumulated during both compatible and incompatible interaction in A. thaliana when challenged with an Oomycete, Peronospora parasitica. If the results exemplified above are interpreted, in incompatible interactions, phytoalexin

accumulation limits or stops pathogen growth, thereby conferring resistance to the plant (Figure 1). In compatible interactions, the pathogen apparently, either tolerates the accumulated phytoalexins, detoxifies them, suppresses phytoalexin accumulation, and/or avoids eliciting phytoalexin production (Mansfield, 1982). Gene manipulation for resistance Phytoalexin definition described above does not include any criteria that would allow discrimination between a role for phytoalexins in defence versus just a response to stress. In order to evaluate the importance of phytoalexins in defence the following criteria can be used: 1. The restriction of the pathogen development must be associated with phytoalexin production, 2. Phytoalexins must accumulate to antimicrobial levels at the infection site in resistant plants or cultivars that could result the cessation of the pathogen growth, 3. There must be strong evidence that the phytoalexins have vital importance in resistance, and absence of these compounds would result enhanced susceptibility (Hammerschmidt, 1999). The first two criteria are easy to satisfy through direct observation of pathogen development in relation to phytoalexin accumulation. The third criterion is the most difficult and complicated one that requires detailed analysis as exemplified below. A central part of evaluating the role of phytoalexins in resistance has been to manipulate phytoalexin accumulation synthesis with inhibitors and enhancers of phytoalexin synthesis and then to determine whether the degree of resistance is altered thereby. If phytoalexin accumulation makes a significant contribution to resistance, cultivars that are normally susceptible to a virulent race of pathogen would be expected to become more resistant, and cultivars that are resistant would become more susceptible to infection, according to whether the phytoalexin content is increased or decreased respectively. In order to find out the role of phytoalexins in resistance, a few elegant models have been studied. The gene for biosynthesis of stillbene phytoalexins has been transferred from Vitis vinifera (grapevine) to tobacco. In response to inoculation with Botrytis cinerea, the transformed plants accumulate mRNA for stilbene synthase, the enzyme specifically required for synthesis of the stilbene phytoalexin esveratrol, around the infection site. They also demonstrate an enhanced resistance to the pathogen that corresponds with accumulation of resveratrol to fungitoxic concentrations (Hain et al., 1993). This affords powerful evidence of the role of phytoalexin accumulation in disease resistance. Glazebrook and Ausubel (1994) and Glazebrook et al. (1997) isolated three phytoalexin-deficient (pad) mutants of A. thaliana accession Col-0 to help elucidate the role(s) of phytoalexins in plant-pathogen interactions. Infection by P. syringae induced the A.thaliana phytoalexin, camalexin, in the pad1, pad2 and pad3 mutants to 30, 10 and <1% (undetectable) of thelevel in wild-type plants, respectively. None of pad mutants was compromised in their ability to resist infection by P. syringae strains carrying avirulence (avr) genes. However pad1 and pad2 exhibited enhanced susceptibility to virulent strains. In contrast, growth of these strains in the pad3 mutant was not significantly different from that in wild-type accession providing evidence that camalexin does not have an important role in protecting the plants from avirulent pathogens but might have a role on restricting the virulent pathogens. Similar results obtained using an Oomycete P. parasitica in a few wild-type accessions and the same mutants (Mert-Trk et al., 1998; Mert-Trk, 2001). Genetic manipulation of secondary metabolites is an area of molecular biology that remains in its infancy. More detailed understanding of the enzymology of phytoalexin synthesis is needed (Mansfield, 1999). Dixon et al. (1996) pointed out that attempts to engineer metabolic pathways may give unexpected results, revealing fundamentally redundant enzyme systems, metabolic channelling, or unexpected translocational control mechanisms. A continued research focus on phytoalexins is therefore, likely to yield data on fundamental aspects of plant metabolism as well as being of indirect benefit to the development of disease control strategies. e.g. gene-for-gene interactions Conclusions Phytoalexins are only one components of the complex mechanisms for disease resistance in plants. Studies on phytoalexins alone have contributed a great deal to plant biochemistry and molecular biology. As they accumulate both in susceptible and resistant plants, the real question is that whether they are contributors to

defence or just the end product of pathogen- or stress-induced metabolism. More conclusive approaches could be used to answer the question. One of them could be to generate phytoalexin biosynthetic mutants that no longer synthesize phytoalexins, then to assess them whether phytoalexin deficiency causes enhanced susceptibility. There are two extremely critical points here that should be kept in mind. This approach should include genetical analyses using a system in which the biochemical and physiological evidence argues strongly in favour of a key role forphytoalexins in resistance. Second point is that the plants must be evaluated for changes in other defences that may compensate for the loss of phytoalexin production. As a result of advances in molecular biology, much better view of the role of phytoalexins in defence has established, though we do still not know for certain whether phytoalexin are contributors for defence. Clearly future studies on these compounds will allow us to understand and evaluate plantpathogen interactions as well as provide new approaches to disease control. All affords in molecular biology and biotechnology is to introduce new approaches into disease control for more friendly environment.