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Pre-fertilization: Reproductive Growth and Development 17 K.V Krishnamurthy Abstract This chapter deals with details on anther and male gametophytic development, ovule and female gametophytic development, events leading to double fertilization, pollen germination and pollen tube and syngamy and triple fusion Since basic embryological developmental details are already detailed in earlier literature, attention is focused only on recent data, particularly molecular data pertaining to these aspects Special attention has been given to genetic control of anther tapetum, endothecium and anther dehiscence, microsporogenesis, microgametogenesis, chalazal behaviour and function and female gametophytic development The importance of cell cycle events in syngamy and triple fusion is highlighted Keywords Anther dehiscence • Chalaza • Embryo sac mutants • Endothecium • Female gametophyte • Male gametophyte • Ovule • Pollen tube • Syngamy • Tapetum • Triple fusion 17.1 Introduction The angiosperm flower typically has four whorls of lateral organs: sepals, petals, stamens and carpels The outer whorls of sepals and petals are sterile and often accessory functions in repro- K.V Krishnamurthy (*) Center for Pharmaceutics, Pharmacognosy and Pharmacology, School of Life Sciences, Institute of Trans-Disciplinary Health Science and Technology (IHST), Bangalore, Karnataka, India e-mail: kvkbdu@yahoo.co.in duction, while the inner whorls of stamens and carpels, respectively, are the male and female reproductive organs producing the male and female gametophytes and gametes There is great variation in the number of stamens from zero in female flowers to one to many depending on the plant species The stamens are free, fused to one another variously to form one to many bundles or attached to the petals or to the carpels Each stamen typically has a stalk (filament) and an anther, the two being attached to each other by a connective Staminal nectaries may be present on the filaments or on the anthers of several species of B Bahadur et al (eds.), Plant Biology and Biotechnology: Volume I: Plant Diversity, Organization, Function and Improvement, DOI 10.1007/978-81-322-2286-6_17, © Springer India 2015 409 K.V Krishnamurthy 410 unrelated families (Chaturvedi and Bahadur 1985) The number of carpels ranges from one to many, free from one another (apocarpous) or fused (syncarpous) to form the gynoecium (or pistil) A typical gynoecium has a basal ovary bearing ovules on special placental tissue (of various types), an apically situated style and a stigma at the tip of the style There is great variation in the size, shape and number of style and stigma depending on the taxon 17.2 Anther and Male Gametophyte The anther is the actual male sexual region of the stamen The term microsporangium is often used as a synonym of anther, but the former term has a much wider connotation and also represents the homologue of the microspore-producing structures of other vascular groups, particularly the pteridophytes (Swamy and Krishnamurthy 1980; Krishnamurthy 2015) Though there are a number of similar developmental features between the anther and the microsporangium of other vascular plants, the male gametophytic organization and behaviour are significantly different The gametophytic cycle in angiosperms shows extreme abbreviation in time and space, and the male gametophyte or pollen is often composed of just two cells, a vegetative cell and a generative cell Anther and pollen development is a critical phase in the life cycle of the angiosperms, and it involves precisely controlled cellular processes including cell division, cell differentiation and cell death due to diverse range of genes and their interaction (Sanders et al 1999; McCormick 2004; Scott et al 2004; Ma 2005) A typical anther is tetrasporangiate although uni-, bi- and octa-sporangiate conditions are also known; these sporangia coalesce to form two sacs or thecae in tetrasporangiate taxa and one in uni- and bi-sporangiate taxa, containing the pollen grains The microsporangia are surrounded by an epidermal layer followed on the inside by the wall layers; the latter are made up of an endothecium, middle layers and a tapetum covering the sporangial locule (Fig 17.1) The anther primordium in transectional view is almost squarish to rectangular and is made of homogeneous parenchymatous tissue, covered by an epidermal layer The archesporial tissue differentiates as a single or a group of two to a few adjacently located cells in the hypodermal position at the four corners of the anther primordium This tissue, in fact, extends vertically from base to the apex of the sporangium The cells of this tissue are distinct from the rest of the anther tissue by their larger size and greater avidity for nuclear and cytoplasmic stains The archesporial cells divide periclinally to form outer primary parietal cells and inner primary sporogenous cells Both these may undergo further periclinal (and a few anticlinal) divisions to respectively form the wall layers and the sporogenous cells (Fig 17.1); rarely the latter directly function as sporogenous cells Based on variations in anther wall development and the number of wall layers present, four types are recognized by Davis (1966): basic, dicot, monocot and reduced types One of the earliest genes required for cell division and differentiation in the anther is the SPOROCYTELESS (SPL)/NOZZLE (NZZ) gene (Schiefthaler et al 1999; Yang et al 1999) In the spl/nzz mutant, archesporial initiation occurs normally, but male sporocyte differentiation is halted and anther development fails to continue The mutant genes of EXTRA SPOROGENOUS CELLS (EXS)/EXCESS MICROSPOROCYTES1 (EMS1) alter the number of archesporial cells Two other genes SOMATIC EMBRYOGENESIS RECEPTORLIKE KINASE1 (SERK1) and SERK2 also have redundant functions during the earlier stages of anther development and, when mutated, result in more sporogenous cells (Albrecht et al 2005; Colcombet et al 2005) 17.2.1 Endothecium The endothecium forms a single layer of hypodermal wall tissue; occasionally, more than one layer may be present in some taxa or may be totally absent as in cleistogamous flowers, aquatic plants and extreme saprophytes The cells of 17 Pre-fertilization: Reproductive Growth and Development 411 Fig 17.1 (a–t), (a–n) Trachyspermum ammi, (o–t) Cuminum cyminum Microsporangium (a, c, e, f, j, k, m) Outline diagrams for (b, d, f, h, j, l) and (n), respectively, showing development of anther (b, d, f, h, j, l, n) Enlargements of portions marked X, X1, X2, X3, X4, X5 and X6 in (a, c, e, g, i, k) and (m), respectively (o, p) Endothecial cells showing thickenings (from whole mounts) (q, r) lateral and surface views of endothecial thickenings (s) Outline diagram of mature anther (t, s) (t) Same, enlargement of portion marked (Sehgal 1965) endothecium are often radially elongated and develop special banded thickening in the inner tangential walls and rarely on radial walls also when the sporangium fully matures (Fig 17.1) The thickening material is not callose but an α-cellulose; in some it may be slightly lignified Transcriptional activity is required for the differ- entiation of endothecium as is evident from the localization of poly(A)-RNA in rice microsporangia by in situ hybridization using [3H] poly(U) as a probe (Raghavan 2000) Just before meiosis poly(A)-RNA concentration decreases sharply in the epidermis and middle layers, a large amount of this is retained in the endothecium Even after 412 the completion of meiosis in the microspore mother cell, some amount of poly(A)-RNA is retained in the endothecium In rice and wheat anthers, the histone H3 gene also activates the endothelial differentiation, particularly in the wild-type and transgenic rice; however, the mechanism of this differentiation is not yet clear The importance of endothecium in anther dehiscence and the way in which the latter occurs are detailed on a subsequent page of this article 17.2.2 Tapetum As already stated, the innermost wall layer of the microsporangium is the tapetum To start with, it borders on the sporogenous cells, and because of its strategic position between the other wall layers and the sporogenous cells, it assumes great significance and importance Although it is found as a single layer all around the sporogenous tissue, it has been shown to have a dual origin (Fig 17.2) The tapetal cells towards the outer sector of the microsporangium are derived from the primary parietal tissue, while those towards the centre of the anther are derived from the connective tissue Although evidences of dual origin Fig 17.2 Development of anther (1–4) to show dual origin of anther tapetum Single-hatched portion of the anther tapetum is of parietal origin, while double-hatched portion is derived from the connective tissue (Periasamy and Swamy 1966) K.V Krishnamurthy of tapetum are lost eventually and become a homogeneous layer in many taxa, there are differences in cell size, shape, number of cell layers, nuclear size, shape and ploidy or time of differentiation, etc between proximal and distal tapeta (Periasamy and Swamy 1966) Two distinct types of tapeta are known in angiosperms: (1) glandular, secretory or parietal tapetum in which the cells retain their walls and persist in situ without much change in shape and position until they perish by programmed cell death (PCD) (Fig 17.1) The tapetal PCD, as the PCD seen in many other plant cells, is a highly orchestrated event that occurs synchronously with pollen mitotic division and formation of pollen exine (Sanders et al 1999) It is relatively rapid and shows chromatin condensation, DNA fragmentation and mitochondrial and cytoskeletal disintegration (Papini et al 1999; Love et al 2008); (2) periplasmodial tapetum, in which the cells lose their inner tangential and radial walls due to enzymatic action of the tapetal cells themselves followed by the coalescence of the protoplasts of all tapetal cells to form a viscous fluid that flows into and fills the sporangial cavity all around the developing microspore mother cells The former type is more common 17 Pre-fertilization: Reproductive Growth and Development in dicots, while the latter in the monocots The glandular tapetal cells are richly protoplasmic, and their nuclei are prominent and metabolically active; in some taxa, nuclei increase in number (two to eight), become polyploidal (due to nuclear fusion or endomitosis) or become polytenic (up to 16 times increase in DNA content) Crystals, starch, lipids, mitochondria, Golgi bodies, ER, membrane-bound ribosomes, plastids, etc are reported in the tapetal cells The cell walls are cellulosic The walls of periplasmodial tapetal cells, before the formation of periplasmodium, have more pectin than cellulose The periplasmodium is an organized structure It gets dehydrated before its complete degradation A third type of tapetum is often recognized and is named amoeboid tapetum (some botanists mistakenly call the periplasmodial tapetum as amoeboid tapetum; see Swamy and Krishnamurthy (1980) for discussion on this) In this type, the cells radially elongate conspicuously and protrude into the sporangial cavity, without, however, losing their cell walls This type is associated with some types of male sterility The tapetum has been considered as a nurse as well as a regulatory tissue for the developing male gametophyte Many indirect evidences are there to implicate the tapetal cells as sources of deoxyribosides which would then be used for DNA synthesis by the microspores, although actual transfer of these from tapetal cells could not be directly demonstrated There are circumstantial evidences to indicate that carbohydrates and pollen reserves may result, at least partially, from the transfer of soluble sugars and peptides or amino acids from the tapetal cells In many plants, there is a close correspondence between tapetal disintegration and the appearance of pollen reserves The most important function of the tapetum is to supply pollen wall and pollen coat polymers (Piffanelli et al 1998) The glandular tapetal cells contain in their cytoplasm numerous bodies, often attached to the lipid membrane-bound, electron-dense organelles known as pro-ubisch, pro-sphaeroid or proorbicule bodies The shape of these bodies varies considerably: granular, rod-shaped, star-shaped, circular, perforated 413 disc-like or compound multiperforate platelike They accumulate as ubisch bodies near the plasma membrane before disappearing from inside the cell They are then immediately seen on the exine of the microspores, where they get integrated as sporopollenin (Fig 17.3) Hence, ubisch bodies are often considered as transport forms of sporopollenin The periplasmodial tapetum, after excessive dehydration, gets deposited on the surface of microspores/pollen grains to form tryphine, a complex mixture of lipoidal substances There is also a deposition of pollenkitt Tapetum controls male fertility/sterility through its timely/untimely production of the enzyme callase (=β-1,3-glucanase) In fertile anthers, it is produced by the tapetum when the callose wall around the microspore tetrad needs to be dissolved to release the individual microspores, while in sterile anthers, the enzyme is often produced precociously to dissolve the callose wall around the microspore mother cell before it undergoes meiosis Some tapetum sequences from anther cDNA libraries of Brassica napus and Arabidopsis specify β-1,3-glucanase Genes that encode proteinase inhibitors of β-1,3glucanase action have been isolated from anthers In situ hybridization with [3H] poly(U) has revealed that mRNA accumulation is one of the metabolic activities that prepares tapetal cells for their function Commensurate with this high metabolic activity, the tapetal cells show the activities of a number of genes At least five tapetum-specific mRNAs and two mRNAs that are also seen in other anther tissues (TA series mRNAs) were demonstrated by in situ hybridization and by the use of chimeric gene constructs in transgenic plants even as early as 1990 (Koltunow et al 1990) These mRNAs get accumulated and lost in the same temporal sequence during tapetum ontogeny and have been identified from a cDNA library of tobacco One of these is TA29 whose product is a glycine-rich cell wall protein that is likely to be involved in exine formation Subsequent studies have revealed the expression products of several other genes An Arabidopsis gene, MALE STERILITY2 (MS2) (Wilson et al 2001; Ito and Shinozaki 2002), is expressed in the tapetum, and the K.V Krishnamurthy 414 Fig 17.3 Summary of pollen wall developmental stages (1–7) (sporoderm) ontogeny of Sorghum bicolor Corresponding developmental stages in the anther locule are also mentioned opposite to each figure (Adapted from Christensen et al 1972; Swamy and Krishnamurthy 1980) DEVELOPMENTAL STAGE primary wall cytoplasm primary wall callose Sporogenous mass Melosis Dyad − Early tetrad primexine bacula −exine tectum columella foot layer endexine Late tetrad Earty vacuolate microspore Late vacuolate microspore Engorged pollen grain intine sequence similarity of this gene’s product to a protein that converts fatty acids to fatty alcohols has implicated this gene to pollen exine formation (Aarts et al 1997) Its rice orthologue is DEFECTIVE POLLEN WALL (DPW) (Shi et al 2011) Loss of function of the FACELESS POLLEN1/WAX2/YRE/CER3 gene causes defects in exine; this gene is likely to encode a putative enzyme of unknown function presumably involved in pollen wall formation (Ariizumi et al 2003) The other rice genes important in tapetal function are WAX-DEFICIENT ANTHER1 (WDA1), OsC6 and PERSISTENT TAPETAL CELL1 (PTC1) Fairly recently, Arabidopsis genes encoding the cytochrome P450 enzymes of CYPTO3A2 and CYP704B1 have been shown to be involved in the biosynthesis of sporopollenin (mutants have severe to moderate defects in exine deposition) (Morant et al 2007; Dobritsa et al 2009) De Azevedo Souza et al (2009) have shown that ACYL CoA SYNTHETASE5 (ACoS5) encodes a fatty acyl synthetase that plays a vital role in exine formation and sporopollenin biosynthesis in Arabidopsis; the acos5 mutant is totally male sterile with pollen lacking recognizable exine Genes that co-regulate along with ACoS5 in pollen exine formation in Arabidopsis such as DIHYDROFLAVONOL4-REDUCTASE LIKE1 (DRL1)/TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) (Grienenberger et al 2010) are also very important, as they affect male sterility (Tang et al 2009) DRL1/TKPR1 is involved in flavonoid metabolism and plays a pivotal role in sporopollenin precursor biosynthesis It was also reported recently that the enzymes closely related to chalcone synthase (CHS) encoded by At1gO2050 [LESS ADHESIVE POLLENS (LAP6)/POLYKETIDE SYNTHASEA (PKSA)] and At4g34850 (LAP5/PKSB) catalyses the sequential condensation of a starter acyl-CoA substrate with malonyl-CoA molecules to produce alkylpyrone in vitro (Dobritsa et al 2010) PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers, mutants of PKS genes displayed exine defects and a double 17 Pre-fertilization: Reproductive Growth and Development pksa pksb mutant was completely male sterile with no apparent exine; these results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACoS5 and PKSA/B are potential and previously unknown sporopollenin precursors (Kim et al 2010) The other genes which are involved in tapetum development and function are ABORTED MICROSPORES (AMS) (Sorensen et al 2003), the rice orthologue TATETUM DEGENERATION RETARDATION (TDR) (Li et al 2006), TAPETAL DETERMINANT1 (TPD1) (Yang et al 2003), DYSFUNCTIONAL TAPETUM (DYT1) (Zhang et al 2006), the rice orthologue UNDEVELOPED TAPETUM (Jung et al 2005), DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1 (TDF1) (Zhu et al 2008), MYB80 (formerlyMYB103) (Higginson et al 2003; Li et al 2007; Zhang et al 2007), ECERIFERUM1 (CER1) (Shi et al 2011) and MS1 (Wilson et al 2001) TDF1 encodes MYB; tdf1 mutant also shows enlarged tapetum with increased vacuolation (Phan et al 2011) and causes arrest of microspore development Early tapetal initiation is affected by the downstream genes EXTRA SPOROGENOUS CELLS (EXS)/EXCESS MICROSPOROCYTES1 (EMS1) (Cannales et al 2002; Zhao et al 2002) and TPD1 Mutants in these genes have an absence of tapetal and middle layers Mutations in SERK1 and SERK2 genes result in the lack of a tapetal layer MYB33 and MYB65 also act redundantly to facilitate tapetal development around meiosis stage; it has been shown that the expression of MYB33 is regulated by miRNAs (Millar and Gübler 2005) These genes are not affected in the dyt1 mutant indicating that they are upstream of DYT1 (Zhang et al 2006) In the dyt1 mutant, tapetum occurs (also meiosis), but tapetum development is abnormal with enlarged vacuoles in its cells DYT1 (by encoding basichelix-loop-helix proteins) has been proposed to be involved in the regulation of many tapetal genes, either directly or indirectly, including AMS and MS1 (Zhang et al 2006) The ams (its wild gene AMS also encodes basic-helix-loophelix proteins) mutant has premature tapetal degeneration because of its abnormally enlarged and vacuolated cells 415 Detailed studies have been done on the role of MS1 gene in tapetal development and pollen wall biosynthesis (Yang et al 2007) Early events in anther development in ms1 mutant are normal and that the MS1 acts, through encoding PHD transcription factors, late in pollen development after tapetal initiation and is downstream of DYT1 (Zhang et al 2006) MS1 coordinates the expression of late genes associated with pollen wall formation and which are involved in the biosynthesis of components of the phenyl-propanoid pathway, long-chain fatty acids and phenolics, which are required for sporopollenin biosynthesis In the ms1 mutant, tapetal PCD does not occur, but tapetal degeneration occurs by necrosis (VizcayBarrena and Wilson 2006); there is also downregulation in the expression of a member of cys proteases in ms1 mutants These proteases are likely to be critical to the progression of PCD, and in their absence, possibly in association with a lack of tapetal secretion, PCD does not occur MS1 also controls the synthesis of pollen coat (oleoresin gene family, lipid transfer proteins or LTPs, ACP lipids and phenyl-propanoid pathway); it does not directly regulate genes associated with pollen wall biosynthesis (due to its timing of expression) but acts via one or a number of additional transcriptional factors (TFs) including MYB99 and two NAM genes that contain a conserved NAC domain (Yang et al 2007) Based on an analysis of transcript levels within tdf1 and ams mutants, Zhu et al (2008) suggested that TDF1 functions upstream of AMS and that AMS is upstream of MYB80 Xu et al (2010) identified 13 genes as direct targets of AMS, but MYB80 was not among them Transcript levels of MS1, MS2 and A6 are downregulated in the MYB80 mutant, suggesting that they act downstream of myb80 It is not known if the three genes are directly or indirectly regulated by MYB80 MYB80 is recently shown (Phan et al 2011) to directly target a glyoxal oxidase (GLOX1), a pectin methyl esterase (VANGUARD1) and an A1 aspartic protease (UNDEAD), all of which are expressed in the tapetum and microspores The timing of PCD in tapetum is likely to be regulated by MYB80/UNDEAD system The overall genetic 416 K.V Krishnamurthy Fig 17.4 Successive divisions of microspore mother cell of Lilium regale (Gerassimova-Navashina 1951) regulation of sporopollenin synthesis and pollen exine development is reviewed by Ariizumi and Toriyama (2011) 17.2.3 Microsporogenesis and Microgametogenesis The sporogenous cells either directly or after a few divisions give rise to microspore mother cells (MMCs) The MMCs possess thin cellulosic cell walls with plasmodesmal connections, not only between themselves but also with the tapetal cells Dictyosomes and plastids (without starch grains) are characteristically present in the cells Most DNA synthesis in MMCs is done during premeiotic interphase, but a meager amount is also synthesized during zygotene-pachytene Similarly, active RNA and protein synthesis takes place during premeiotic stage with a fall during meiotic prophase There is a decline in ribosomal population after the initiation of meiosis, but the population is restored after homotypic division There is also a reorganization of mitochondria and plastids in the microspore, as they are partly degraded during meiosis Just at the onset of meiosis in MMCs, a callose wall is deposited inner to the original cellulosic wall Any irregularity in callose deposition/metabolism results in male sterility Callose deposition starts on the walls of MMCs close to tapetum and gradually extends to the more centrally located cells of the anther Initially, the callose wall is incomplete leaving many gaps in the wall through which massive cytoplasmic channels between adjacent MMCs (but not with tapetum cells) are established These channels reach their maximum development during zygotene-pachytene and help establishing near synchronicity in meiosis in all MMCs of a sporangium Callose deposition is considered as a necessary prerequisite for meiotic induction and continuance (Krishnamurthy 1977, 2015) Callose is highly impervious to most molecules and thus is a highly isolating and insulating material The plasmodesmal connections are sealed off towards the end of metaphase I in taxa with successive division and at anaphase II in plants with simultaneous division Two types of meiotic division are known in MMCs, either of which results in the formation of a tetrad of four microspores In successive division, a centrifugally extending cell plate and then a wall are promptly laid down between the daughter nuclei at the end of each of the two divisions (Fig 17.4) In the simultaneous division, the separation of all four microspore nuclei is 17 Pre-fertilization: Reproductive Growth and Development 417 Fig 17.5 Trachyspermum ammi Microsporogenesis and male gametophyte; (a–j) Simultaneous meiotic division in microspore mother cell leading to tetrad formation; (k–n) Uninucleate microspore (o–p) Two-celled pollen (q) Three-celled pollen; (r) Palynogram (Sehgal 1965) effected through centripetally extending furrows at the end of the second division (Fig 17.5a–j) The callose wall around the tetrad is heterogeneous and layered The outermost layer is the most well developed Three more concentric layers follow this on the inside distinguished from each other by their variable density The fifth layer is the innermost and the least dense of all It surrounds and isolates the four microspores and cell plates Each microspore is individually surrounded by the primexine Soon after meiosis, callose wall around the microspore tetrad is degraded by β-1,3-glucanase into D-glucose and oligomers of D-glucose of different lengths, which may be used by the microspores for various purposes (such as nutrition and pollen wall formation) As a result of callose degradation, the individual microspores are separated out of the 418 tetrads β-1,3-glucanase is present in low quantities in the tapetum even during meiosis in MMCs, but increases suddenly during late tetrad stage to cause the separation of microspores In some angiosperms, failure of microspores to separate out of the tetrads results in the formation of permanent tetrads or compound pollen grains In some Mimosaceae and Orchidaceae, polyads of 8–32 grains called massulae are formed An extreme case of adherence of all pollen grains of an entire microsporangium is seen in many Asclepiadaceae and the resultant structure is called a pollinium The studies made so far show that both the diploid sporophytic tapetal cells and the haploid gametophytic microspore contribute to pollen wall synthesis (Ariizumi and Toriyama 2011) Exine formation is stated to commence from the late tetrad stage with the laying down of the primexine between the callose wall and the plasma membrane of the microspore (Paxson-Sowders et al 1997) (except at the germinal pore region where it is absent) The microspore just released from the tetrad does not have an exine (the outer wall of the pollen) The primexine is distinguished from the callose by its electron opacity It has a matrix, presumably made up of cellulose, and radially directed rods, the probaculae and profoot layer The deposition of sporopollenin begins immediately after release of microspores from the tetrad, and its source is from the tapetum, as already detailed The characteristic pattern of the sporoderm is determined by features already imprinted in the primexine during the period of enclosure in the tetrad (Blackmore et al 2007) However, a few investigators believe that the initial exine pattern laid down in the microspore is controlled by the plasma membrane and that callose causes this imprinting by acting as a template (and not the primexine) After the first division of the microspore, exine formation is almost complete At later stages of pollen ontogeny, pectocellulosic intine and tryphine are deposited (Piffanelli et al 1998) Intine formation first begins in the vicinity of the germinal aperture(s) and from there spreads all around the microspore; this growth is said to be associated with dictyosome activity in coordination with the K.V Krishnamurthy plasma membrane Thus, intine is programmed entirely by the haploid, male gametophytic genome and is made of pectocellulose, while the exine is organized both through tapetal inputs and microspore activity Under typical conditions, the microspore nucleus occupies a central position, while the cytoplasm has many small vacuoles spread almost evenly (Fig 17.5k–n) Just before division, the nucleus moves towards a side that is generally opposite to the furrows Mitochondria and plastids are displaced to the cytoplasm opposite to the nucleus During interphase, active ribosomal RNA synthesis takes place A conspicuously large vacuole appears in the cytoplasm opposite to the nucleus The nucleus then divides followed by a curved callose wall to result in a small lens-shaped daughter cell (appearing spindle shaped in cross-sectional view) called the generative cell (GC) and a conspicuously larger cell called vegetative cell (VC) (Fig 17.5o, p) Thus, the division is asymmetric The callose wall separating the GC from VC is highly transitional and is retained only for about 10–20 h GC soon gets pinched off from the microspore wall and becomes embedded in the cytoplasm of the VC, by which time its callose wall is also lost This may or may not be accompanied by a change of shape of the GC This separation is effected by the growth of callose wall in between the plasma membrane of the GC and the intine of pollen grain The new location of GC obviously provides a new environment for interaction between GC and VC At this stage, the pollen is said to be mature in most taxa The GC is surrounded by a double membrane, by a distinct cellulosic wall or by the retention of the original callose wall depending on the species The GC is less dense due to very poor or even no RNA and proteins Minute vacuoles filled with water or lipid materials are also present The DNA content of its nucleus is very high (rises to 2C level), but the nucleolus is not very conspicuous Axial microtubules have been recorded and these are important in controlling the shape of the GC However, there is some disagreement regarding the cytoplasmic organelles of the GC, probably because of species-dependent variations Mitochondria, 31 Alien Crop Resources and Underutilized Species for Food and Nutritional Security of India Disclaimer The views and opinions expressed in this book chapter are 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largest global primary producers, are a potential source of bioactive compounds They are unique in producing superfine chemicals that can be used in various industrial sectors like pharmaceuticals, nutraceuticals and cosmeceuticals The chapter is intended to provide an insight to two of the most important pigments obtained from them, phycobiliproteins and carotenoids having species specificity which can be used as a chemotaxonomic marker Their unique structural properties play a crucial role in their biological functions The water-soluble phycobiliproteins are used as fluorescent tags in flow cytometry and immunochemistry, while liposoluble carotenoids are potential alternatives to synthetic dyes in the food industry Keywords Microalgae • Phycobiliproteins • Carotenoids • Fluorescence • Applications 32.1 T Ghosh • C Paliwal • R Maurya • S Mishra (*) Academy of Scientific and Innovative Research, New Delhi, India Discipline of Salt & Marine Chemicals, CSIRCentral Salt and Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat, India e-mail: smishra@csmcri.org Introduction Photosynthesis is an important biochemical reaction responsible for meeting our energy demands directly or indirectly The solar energy received on earth is converted into chemical energy by means of photosynthesis, which is then stored in various forms Algae, either unicellular or filamentous, are one of the most primitive photosynthetic organisms found in both freshwater and marine habitats They are subdivided as macroalgae (seaweeds) and microalgae Microalgae are global primary producers which contribute from one-third to more than B Bahadur et al (eds.), Plant Biology and Biotechnology: Volume I: Plant Diversity, Organization, Function and Improvement, DOI 10.1007/978-81-322-2286-6_32, © Springer India 2015 777 T Ghosh et al 778 Table 32.1 General composition of different algae (% of dry matter) Protein Strain 50–56 Scenedesmus obliquus 22 Botryococcus braunii 8–18 Scenedesmus dimorphus 48 Chlamydomonas reinhardtii 51–58 Chlorella vulgaris 10–20 Chlorella protothecoides 6–20 Spirogyra sp 55–65 Dunaliella tertiolecta 57 Dunaliella salina 39–61 Euglena gracilis 28–45 Prymnesium parvum 52 Tetraselmis maculata 28–39 Porphyridium cruentum 46–63 Spirulina platensis 60–71 Spirulina maxima 63 Synechococcus sp 43–56 Anabaena cylindrica Source: Adapted from Spolaore et al (2006) half of the total primary productivity (Van Den Hoek et al 1995; Miyamoto 1997; Guschina and Harwood 2006) Because of their high growth rates and ability to mitigate CO2 from the environment and utilize non-arable land for their cultivation, they are considered as potential energy feedstock for their utilization in biofuel (biodiesel, bioethanol and biogas etc) production Apart from being an energy feedstock, microalgae are a great store of many different biomolecules such as polyunsaturated fatty acids (PUFAs), sterols, pigments, enzymes, vitamins, minerals, proteins and carbohydrates which are beneficial both economically and medically The general composition of the algae in terms of carbohydrates, lipids, nucleic acids and proteins is provided in Table 32.1 They are able to potentially accumulate up to 50 % of their dry weight as carbohydrates, primarily in the form of starch, glucose, cellulose or hemicelluloses or polysaccharides of various kinds (Ho et al 2012; Yen et al 2013) Algal polysaccharides are, to a large extent, sulphated polysaccharides with important medical applications Crude polysaccharide extracts from vari- Carbohydrates 10–17 18 21–52 17 12–17 12–20 33–64 10–15 32 14–18 25–33 15 40–57 8–14 13–16 15 25–30 Lipids 12–14 55–60 16–40 21 14–22 55 11–21 20 14–20 22–38 9–14 4–9 6–7 11 4–7 Nucleic acid 3–6 – – – 4–5 – – – – – 1–2 – – 2–5 3–4.5 – ous microalgae such as Chlorella vulgaris, Chlorella stigmatophora, Scenedesmus quadricauda and Phaeodactylum tricornutum have anti-inflammatory, immunomodulatory and antioxidant properties (Guzman et al 2003; Mohamed 2008) Microalgae synthesize ω-3 and ω-6 fatty acids including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), γ-linoleic acid and arachidonic acid (ARA) essential in maintaining the tissue integrity, which humans are not able to synthesize They have many beneficial properties like they are anti-inflammatory, play a role in brain development, help in functioning of the nervous system and delay ageing Most of these polyunsaturated fatty acids (PUFAs) are used as health supplements, as baby food additives, in therapeutics and as poultry feed (Ahren et al 1983; Cohen and Heimer 1992; Gordon and Ratliff 1992; Borowitzka 1993; Barclay and Zeller 1996; Pulz and Gross 2004; Guedes 2010; De Jesus Raposo et al 2013) Some species of microalgae are also found to be rich in vitamins and industrially important enzymes Porphyridium cruentum is a good source of vitamins C and E as well as provitamin 32 Microalgal Rainbow Colours for Nutraceutical and Pharmaceutical Applications A (Sarrobert and Dermoun 1991) Navicula ostrearia, a diatom, is a rich source of vitamin E (De Jesus Raposo et al 2013) Dunaliella salina, besides being known for β-carotene production, is also a source of thiamine, pyridoxine, riboflavin, nicotinic acid, biotin and tocopherol (Drokova and Popova 1974) Carbonic anhydrase, a crucial enzyme responsible for the conversion of CO2 into bicarbonate ions and carbonic acid, is produced by Isochrysis galbana, Amphidinium carterae and Prorocentrum minimum (Yingying and Changhai 2009; De Jesus Raposo et al 2013) Superoxide dismutase, another enzyme crucial for antioxidant activity in vivo, is produced by Anabaena sp., Porphyridium sp., Phaeodactylum tricornutum and Synechococcus sp (Thepenier et al 1988; Guzman-Murillo et al 2007; De Jesus Raposo et al 2013) We are witnessing a shift of research interests in functional foods obtained from natural sources, which contain additional nutrients and are beneficial to humans There has been felt a need to investigate such potentially important high-value products like antioxidants, anti-inflammatory compounds, natural colouring agents, fluorescent dyes and many others, from natural sources (Eisenreich et al 2004) Microalgae have been exploited for such bioactive compounds for use in pharmaceutical, nutraceutical, food and cosmetic industries From an economic point of view, microalgal cultivation is often preferred for the production of high-value compounds like phycobiliproteins and carotenoids (Spolaore et al 2006; Chu 2012; Markou and Nerantzis 2013) Nevertheless, there still exists a need to improve microalgal cultivation and harvesting technology along with the techniques used for the extraction and purification of the desired molecules (Molina et al 2003) Currently, different food companies are interested in improving their products through substitution of natural products because of larger profit margins compared to conventional food products and their acceptability to the public in general (Hasler 2002; Siro et al 2008) A full description of all these biomolecules would exceed the scope of this chapter which pri- 779 marily details about the various pigments sourced from these organisms Our main focus would be pigments derived from marine microalgae which have potential health and commercial benefits A number of these pigments have antioxidant, antiinflammatory and neuroprotective properties which have been conclusively proved through various in vitro and in vivo studies Apart from these biological properties, they play an important role in diagnostic biosensors and fluorescence analytical techniques Microalgal pigments are broadly classified into two groups: • Water-soluble phycobiliproteins sourced from microalgae as well as macroalgae are used for developing fluorescent markers in conjugation with immunoglobulins and other proteins • Lipid-soluble carotenoids such as astaxanthin, zeaxanthin and ß-carotene from microalgae which serve as provitamins and antioxidants 32.2 Phycobiliproteins Phycobiliproteins are accessory light-harvesting pigments predominantly found in cyanobacteria (blue-green algae), Rhodophyta (red algae), Cryptophyta and Glaucophyta The phycobiliproteins are further classified on the basis of their spectral properties into three major subgroups: phycocyanin, allophycocyanin and phycoerythrin Their composition varies with the species and environmental conditions of the source organism (Chu 2012) Due to their fluorescent properties, they were adopted for use in diverse applications such as fluorescence-activated cell sorting, flow cytometry and histochemistry soon after their introduction as pigmented molecules in 1982 They can also be used as markers for electrophoresis, isoelectric focusing and sizeexclusion chromatography due to their high absorptivity in visible light wavelengths 32.2.1 Structure Phycobiliproteins are composed of apoproteins (α and β subunits) covalently linked to prosthetic 780 T Ghosh et al Fig 32.1 Structure of a phycobilisome groups called phycobilins Phycobilins are openchain, tetrapyrrole chromophores sharing structural similarity with the bile pigment bilirubin (Glazer 1989) The two conserved subunits, α and β, form an (αβ) monomer, which are further aggregated to form trimers (αβ)3 and disc-shaped hexamers (αβ)6 The trimeric and hexameric structures form the functional units of PE and PC In a complete LHC, also termed as a phycobilisome, the central core is occupied with rods of APC joined to disc-shaped hexameric PC and PE which extend outwards as antennae (Fig 32.1) The light energy is captured by PE and is transferred to chlorophyll for further reaction via PC and APC The absorption maxima vary from 562 to 568 nm for C-PE, 615 to 620 nm for C-PC and 650 to 652 nm for APC (MacColl 1998) (Table 32.2) 32.2.2 Extraction and Purification of Phycobiliproteins The extraction of phycobiliproteins chiefly involves cell disruption in a buffered environment after which the crude extract is either centrifuged or filtered to remove cellular debris Cell disruption is done through ultrasonication, freeze-thaw cycles using liquid nitrogen, cavitation using nitrogen gas, osmotic shock, enzymatic treatments or high-pressure homogenization (Table 32.3) Wet biomass is directly utilized for the extraction of these proteins as high-temperature drying usually results in a lower-quality product or a lower yield Usually, 0.05 or 0.1 M phosphate buffer pH 7.0 or 7.2 is used as the extraction buffer although 0.5 M ammonium sulphate is also used Purification of these proteins is usually done using ammonium sulphate precipitation, polyethylene glycol precipitation, ion-exchange or sizeexclusion chromatography, expanded bed chromatography or membrane filtration to get their purified forms More often than not, a combination of these techniques is used to reach the desired purity level and the source organism Drying is usually performed using lyophilization which prevents denaturation of the pigment The measure of purification is determined by calculating the purity ratio, a ratio of the absorbance of the particular phycobiliprotein at its absorption maxima to that of aromatic amino acids in all proteins at 280 nm For example, the purity ratio 32 781 Microalgal Rainbow Colours for Nutraceutical and Pharmaceutical Applications Table 32.2 Spectral and physical properties of cyanobacterial phycobiliproteins Phycobiliproteins C-phycocyanin C-phycoerythrin Allophycocyanin Absorbance maxima (nm) 615 566 652 Fluorescence emission (nm) 647 617 660 Molecular weight (kDa) 108 55 100 Absorptivity (L g−1 cm−1) 7.0 8.0 7.3 Molar absorptivity (M-cm)−1 1.54 0.44 0.73 Table 32.3 Different methodologies adopted for the extraction of phycobiliproteins Extraction method Freeze-thaw and sonication Phycobiliprotein Phycocyanin Name of species Spirulina platensis High-pressure homogenization Phycocyanin Spirulina platensis Freeze-thaw Phycoerythrin, phycocyanin, allophycocyanin Spirulina platensis, Phormidium sp A27DM, Lyngbya sp A09DM, Halomicronema sp A32DM, Pseudanabaena tenuis, Spirulina fusiformis, Arthronema africanum, Calothrix sp., Oscillatoria quadripunctulata, Pseudanabaena sp Sonication Phycoerythrin Variable speed stirring Phycocyanin Nitrogen cavitation Phycobiliproteins Cyanosarcina sp SK40, Phormidium sp PD40-1, Scytonema sp TP40, Leptolyngbya sp KC45 Anabaena marina ATCC 33047 Synechococcus sp Lysozyme treatment Phycocyanin Synechococcus sp of C-PE is calculated by A568/A280 and for C-PC using A620/A280 The absorbance values are considered within a range of 0.05–1 at the absorptive maxima of the phycobiliprotein (Bennett and Bogorad 1973) A ratio greater than is generally considered food grade, while a ratio greater than is considered as analytical grade purity The purified forms of the proteins are generally stable in phosphate buffer pH 7.0 or 7.2 or in ammonium sulphate suspensions The latter are usually dialyzed against the corresponding buffer before use They are stored in temperatures 4–10 °C in the dark to reduce the effects of light References Zhang and Chen (1999) Patel et al (2004), Song et al (2013) and Seo et al (2013) Minkova et al (2007), SantiagoSantos et al (2004), Soni et al (2010), Minkova et al (2007), Mishra et al (2008), Su et al (2010), Cano-Europa et al (2010), Parmar et al (2011) and Mishra et al (2011) Pumas et al (2011) Ramos et al (2010) Viskari and Colyer (2003) Gupta and Sainis (2010) 32.2.3 Applications of Phycobiliproteins 32.2.3.1 As Food Colourants Natural colouring agents have always held an upper hand when it comes to the food industry Due to the toxic nature of synthetic colourants and the necessity of colour additives for food processing, there is an increased awareness and curiosity for natural options in this field However, studies are still underway for the stability of such proteins in pH ranges used in commercial food manufacturing industries worldwide PBPs are used as natural food colourants in chewing gums, T Ghosh et al 782 jellies, ice creams and fermented milk products since many of the synthetic dyes used globally are thought to be possible carcinogens Their other advantages include their intense colours and high solubility in water (Santiago-Santos et al 2004; Chakdar et al 2012) A lower stability to temperature and light has not deterred the food processing industry to use C-PC as an alternative to synthetic dyes such as gardenia and indigo A study reported that C-PC was insoluble in acidic solutions (pH 3) and denatures at temperatures above 45 °C (Jespersen et al 2005) Additionally, the fluorescent properties of PE have been exploited to produce transparent lollipops, cake decorations and soft drinks and alcoholic beverages that fluoresce at pH 5–6 These special effects were tried out to increase the marketability of the respective food items (Dufosse et al 2005) Although still not approved for use in the USA and European Union (EU), the US Food and Drug Administration (US FDA) has been approached by Desert Lake Technologies in 2012 for grant of generally recognized as safe (GRAS) status to C-PC (CyaninPlus™) developed by them (FDA, GRAS 2012) 32.2.3.2 As Pharmaceutical Agents PBPs have been recognized as beneficial pharmaceutical agents since many years, and the fact has been reliably established through studies Oriental cuisine and medicine have traditionally been rich in microalgae since ancient times, but it is only now that their beneficial effects are being investigated scientifically (Bocanegra et al 2009) The current total market value of PBP products has been estimated to be US $60 million (Borowitzka 2013) The nutritional and therapeutic aspects of Spirulina, a blue-green algae, have been critically reviewed which have proved that the beneficial aspects of the cyanobacteria can be attributed to its C-PC content among other things (Kay and Barton 1991; Mishra 2006) 32.2.3.3 As Antioxidants The antioxidant properties of PC have been well documented over the years According to published research, C-PC successfully reduced lipid peroxidation and oxidative haemolysis in normal human erythrocytes induced by a free-radical generator, AAPH C-PC extract from Aphanizomenon flos-aquae, a cyanobacterium, was also found to significantly inhibit lipid peroxidation in blood plasma by Cu+2 (Benedetti et al 2004) In vitro studies have established C-PC as an antioxidant, anti-inflammatory, neuroprotective, nephroprotective and hepatoprotective agent (Romay et al 1998; Farooq et al 2004; Mishra 2006; Sekar and Chandramohan 2008) Radical scavenging activity of C-PC includes scavenging peroxyl, peroxynitrite and hydroxyl free radicals while preventing or inhibiting lipid peroxidation and DNA damage (Bhat and Madhyastha 2001; Bermejo et al 2008) C-PC has been demonstrated to significantly reduce hippocampal cell death in gerbils and rats (Thaakur and Sravanthi 2010; Penton-Rol et al 2011), reduces necrosis and inflammation in hepatic cells (Gonzalez et al 2003; Sekar and Chandramohan 2008; Kuriakose and Kurup 2010) and decreases Kupffer cell phagocytosis (Remirez et al 2002) C-PE from Pseudanabaena tenuis was examined for its antioxidant ability in mice model fed with mercury and found to reduce the extent of damage in all animals (Cano-Europa et al 2010) 32.2.3.4 As Anti-inflammatory Molecules C-PC was also analysed as an anti-inflammant in human models where its ability to effectively inhibit the activity of cyclooxygenase-2, an enzyme involved in the process of inflammation, was studied It was found that although C-PC effectively inhibited the said enzyme, reduced PC or the isolated PCBs were poor inhibitors without being selective for the enzyme The results suggest that the apoprotein of C-PC may have an important role to play in the anti-inflammatory activity of C-PC (Reddy et al 2000) Another study analysed the in vivo effect of excessive C-PE dosage in test mice to evaluate the potential risks due to overdosage It was found that C-PE had no deleterious effect on the body weight, food intake or toxicity signs even at a dosage of 2,000 mg kg−1 body weight C-PE This is a significant finding for adopting a C-PE based 32 Microalgal Rainbow Colours for Nutraceutical and Pharmaceutical Applications treatment approach since it indicates no negative health effects through overdosage (Soni et al 2010) 32.2.3.5 As Anticancer Agents The anticancer activity of C-PC was studied using human chronic myeloid leukaemia cell line K562 It was observed that as little as a 50 μM dose decreased the proliferation of K562 cells by 49 % for 48 h (Subhashini et al 2004) Also, C-PC induced apoptosis in prostate cancer (LNCaP) cell line by diminishing the required dosage of topotecan, an anticancer medication which frequently causes adverse side reactions in patients (Gantar et al 2012) In another study involving human hepatoma cell line (HepG2), C-PC led to a reduction in the proliferation of the cells with the highest reduction observed at a concentration of μg/ml C-PC along with a loss of nuclear entities due to fragmentation (Basha et al 2008) A separate study has examined the healing effect of C-PC on workers exposed to nuclear radiations in a nuclear power plant The study was carried out as part of a publication on nuclear power plant operations, safety and environment It was found that C-PC has the ability to influence repair of damaged DNA, essential for the preservation of genomic integrity However, the protein also showed DNA lesion in subjects exposed to high doses of radiation; the lesions were not found to be persistent This may be attributed to the adaptive phenomena due to the chronic adaptation exposure Although the results are promising, the authors categorically state that the study should be treated as pilot one with the need for further experiments to prove conclusively the role played by PBP such as PC in DNA repair mechanisms (Stankova et al 2011) The studies firmly establish the anticancer properties of C-PC that might open up an exciting avenue for medical treatments for various life-threatening cancers Although the findings are rather sporadic instead of being coherently directed towards a particular type of cancer, the promising results are sure to encourage researchers to focus more on specific types of cancer 783 32.2.3.6 As Fluorescing Molecules The fluorescence properties of PBP have played an important role in the development of various fluorescence-based techniques including fluorescence-activated cell sorting (FACS), flow cytometry, protein-protein conjugation and fluorescence immunoassays and fluorescence microscopy (Mishra 2006) There are many reasons which can explain the advantages of using PBP as fluorescent molecules, such as (1) low interference by other molecules due to absorption and emission at far red end of spectrum, (2) a large Stokes shift of 80 nm or more which minimizes noise due to other phenomena, (3) high solubility in water leading to minimal side reactions, (4) quantum yield independent of pH and (5) protection from quenching by other biological molecules (Kronick and Grossman 1983) An excellent overview of the relevant properties of PBPs has been provided in Glazer (1994) The isoelectric points of the PBP range from 4.7 to 5.3 32.2.3.7 Bioconjugates Conjugation of proteins with PBP has attracted much interest due to their highly sensitive detection characteristics and multiparameter detection A recent US patent application has claimed a process to develop fluorescent kits using PBP and chemical dyes attached together to take advantage of the intermolecular energy transfer phenomena (Mao et al 2012) However, the conjugation studies and applications are not new The utility of such conjugated molecules for cell cytometry applications and diagnostic procedures was recognized long time ago (Stryer and Glazer 1985) The only limiting factor has been the molecular weight of the PBP (200 kDa) which hinders their diffusion into cells of interest and hence limits their applicability to antibody conjugates for flow cytometry and enzyme-linked immunosorbent assays (Giepmans et al 2006) Another example of a conjugated PBP with a suitable dye molecule utilizes R-PE and compares the fluorescence of the pair to that of native R-PE to assess energy transfer from the PBP to the dye The transfer efficiency was found to be >99 % The conjugate was used to label streptavidin that retained the fluorescence properties and was T Ghosh et al 784 useful in flow cytometry applications (Diwu et al 2012) The fluorescence properties of B-PE were studied in a nonpolar environment using AOT (sodium bis-(2-ethylhexyl)sulphosuccinate)/ water/isooctane micro-emulsions AOT is an anionic surfactant that can solubilize small quantities of water in various nonpolar solvents Results indicated that the stability of B-PE in water droplets inside the emulsion is enhanced than the protein that is in the aqueous state It may be that the protein inside the water droplet retains the same configuration and hence its fluorescence properties, but the chromophores are more protected inside the emulsified environment (Bermejo et al 2003) PBPs are extremely amenable to bioconjugation and have bright chromophores These factors have together contributed to their being used as fluorophores conjugated to various other molecules using standard chemistry The only drawbacks of the process are that the reactions should be suitable for a biological origin molecule and not disturb the original configuration to avoid a loss of fluorescence Although limited reports are available for PBP as nanomaterial, commercial applications for the few discovered bioconjugates have already been in use for some time Commercial ventures are already manufacturing and marketing PBP-conjugated fluorescent dyes and antibodies for flow cytometry and immunolabelling, respectively (Sapsford et al 2013) 32.3 Carotenoids Carotenoids are composed of more than 600 natural lipid-soluble pigments with their colour ranging from yellow to red and are found predominantly in plants (Takaichi 2000; Kleinegris et al 2010) The structures of carotenoids differ in cyclization (one or both ends of the molecule), hydrogenation level and functional groups (Dutta et al 2005) Some carotenoids are found in both plants and algae, while some are limited only to algae (Takaichi 2011) Chemical synthesis is a low-cost method for obtaining high-purity carotenoids, but its major drawback is the non-biological reaction precur- sors/by-products which may have deleterious health effects, and hence, it is suggested to find economical carotenoid production of biological origin The modern tools of bioprocessing and recombinant DNA technology can significantly increase carotenoid production Also, it is important to know that there are several disadvantages associated with the production of carotenoids from food such as complicated extraction and purification process, season fluctuation, limited resources, etc (Asker et al 2012) Carotenoids are isoprenoid compounds made up of 40-carbon (C40) backbone synthesized via head-to-head condensation of two geranylgeranyl diphosphate (GGDP, C20) molecules Naturally occurring carotenoids are generally trans in nature (Dutta et al 2005) They are synthesized in living systems by carotenogenesis pathways, which have been extensively studied in cyanobacteria (Takaichi 2011) Carotenoids exhibit different properties like singlet oxygen quencher, binding affinity for hydrophobic surfaces, antitumor activity, provitamin A activity, anti-inflammatory activity, hepatoprotective activity and antioxidant activity and are also a part of cellular communication, immune-modulation activity such as decrease in UV-induced immune suppression and increase the activity of natural killer cells (van den Berg 1999; Dutta et al 2005; Vílchez et al 2011; Han et al 2012) Carotenoids are mainly classified into two subgroups (Sergio et al 1999): (a) Carotenes: Hydrocarbons consisting of specific end groups, e.g lycopene and ß-carotene (b) Xanthophylls: Oxygenated carotenoids Xanthophylls are further subdivided depending upon the type of functional groups attached: (i) Containing hydroxyl groups, e.g zeaxanthin and lutein (ii) Containing methoxy group, e.g spirilloxanthin (iii) Containing oxo group, e.g echinenone (iv) Containing epoxy group, e.g antheraxanthin Carotenoids can also be classified as primary and secondary carotenoids Among them, primary 32 Microalgal Rainbow Colours for Nutraceutical and Pharmaceutical Applications carotenoids play a crucial role in the photosynthetic organisms, while the secondary carotenoids are not essential for photosynthesis and are localized either in plastoglobules or in cytosolic lipid droplets; they are produced under stress conditions and can be accumulated to high levels (Sasso et al 2012) 32.3.1 Microalgal Sources Different microalgal strains of research interest are Dunaliella salina, Sarcina maxima, Chlorella protothecoides, Chlorella vulgaris and Haematococcus pluvialis for the commercialization of carotenoid production (Lordan et al 2011) The table below depicts different microalgae as sources of carotenoids (Table 32.4) 32.3.2 Factors Affecting Production of Carotenoids The growth conditions and environmental parameters are important parameters that control carot- 785 enoid accumulation in organisms (Walter and Strack 2011) The factors that affect carotenoid production in marine microalgae are described below: Light There are different theories on which photostimulation of carotenoid synthesis depends; one describes high light intensity, and other focuses on high illumination time to cause a rise in carotenoid concentration (Bhosale 2004) Temperature Temperature plays a crucial role in carotenoid production; a decline in thermal conditions from 34 °C to 17 °C caused a 7.5 times increase in α-carotene content in Dunaliella sp (Bhosale 2004) Nutrients Nannochloropsis gaditana deprived of phosphate/sulphur causes an improvement in zeaxanthin concentration due to rapid inhibition of PSII driven by S-limitation that diminishes the primary photosynthetic product formation, i.e NADPH and Fdred which later on caused insufficient ascorbate supply for the Table 32.4 Microalgal sources of different carotenoids Carotenoids α-carotene ß-carotene Lutein Astaxanthin Zeaxanthin Fucoxanthin Canthaxanthin Sources Dunaliella salina Dunaliella salina, Botryococcus braunii, Spirulina platensis, Chlorococcum sp., Synechocystis sp., Parietochloris incisa Muriellopsis sp., Scenedesmus almeriensis, Chlorella protothecoides, Chlorella zofingiensis, Botryococcus braunii, Neospongiococcus gelatinosum, Chlorococcum citriforme Chlamydomonas acidophila, Diacronema vlkianum Haematococcus pluvialis, Botryococcus braunii, Chlorella zofingiensis, Scotiellopsis oocystiformis, Neochloris wimmeri, Diacronema vlkianum, Euglena rubida Dunaliella salina, Spirulina sp., Microcystis aeruginosa, Botryococcus braunii, Chlamydomonas acidophila Phaeodactylum tricornutum, Cylindrotheca closterium, Eustigmatos magnus, Eustigmatos polyphem, Eustigmatos vischeri, Vischeria helvetica, Vischeria punctata, Vischeria stellata Anabaena spp Reference Christaki et al (2013) Del Campo et al (2007), Solovchenko et al (2008) and Ranga Rao et al (2010) Fernández-Sevilla et al (2010), Del Campo et al (2007), Ranga Rao et al (2010), Cuaresma et al (2011) and Durmaz et al (2009) Christaki et al (2013), Ranga Rao et al (2010), Del Campo et al (2004), Orosa et al (2000), Zhang and Lee (1997) and Durmaz et al (2009) Christaki et al (2013), Ranga Rao et al (2010), Sajilata et al (2008) and Cuaresma et al (2011) Kim et al (2012) and Li et al (2012a, b) Shahidi and Brown (1998) T Ghosh et al 786 xanthophyll cycle and hence reduced xanthophyll biosynthesis (Forján et al 2007) Metal ions/salts Addition of ferrous salt increases the hydroxyl radical which, in turn, promotes cellular carotenoid synthesis in Haematococcus pluvialis This method can substitute high light illumination which is costlier and an energy-intensive process Chlorococcum spp has provided similar results in the presence of inorganic salts (Bhosale 2004) 32.3.3 Extraction of Carotenoids The major issue in microalgal biotechnology is the downstream processing where microalgal biomass harvesting remains a prominent research area Experience suggests that effective harvesting technology is completely dependent on strain characteristics (Del Campo et al 2007) There is no defined protocol for the extraction of carotenoids as various factors contribute in the transformation or degradation during their extraction; various precautions like dim light, antioxidants need to be taken to prevent photo-damage and oxidation (Oliver and Palou 2000) Extraction can be performed using organic solvents like hexane, methanol and acetone, but they are not recommended due to their toxicity Green solvents like vegetable oils or supercritical CO2 are more suitable for this process (Wiltshire et al 2000; Macías-Sánchez et al 2008; Guedes et al 2011; Christaki et al 2013) 32.3.4.2 Antioxidant Activity Reactive oxygen species (ROS) include both free radicals and non-radical oxidants which are the most reactive molecular species responsible for DNA, protein and lipid degradation (Pérez-Rodríguez et al 2009) Carotenoids have the ability to scavenge singlet molecular oxygen and peroxyl radicals, which makes them strong antioxidants (Sies and Stahl 2004) These properties help them in preventing chronic and degenerative diseases like cancer A study shows that the risk of colon cancer gets reduced due to the inclusion of ß-carotene in the diet (Vílchez et al 2011) High ß-carotene doses show better CD4 to CD8 lymphocyte ratio (Christaki et al 2013) 32.3.4.3 Membrane Stabilization Researchers have reported mechanical stabilization of liposomal membranes by carotenoids such as zeaxanthin at higher temperatures (Hara et al 1999) They form a complex molecular structure with lipid membranes and control the dynamics and physical properties of lipid membranes, protecting lipid peroxidation (Popova and Andreeva 2013) It was also found that incorporation of carotenoids into membrane decreases their permeability, whereas polar carotenoids on phospholipids mimic as cholesterol and play important role in the modulation of membranes which does not contain cholesterol (Gruszecki and Strzalka 2005) 32.3.5 Practical Applications 32.3.4 Properties of Carotenoids 32.3.4.1 Provitamin A Activity Provitamin A activity is the conversion of provitamin A into vitamin A, whose deficiency leads to premature deaths, particularly among children About 10 % of the natural carotenoids have the ability to get converted into retinol which has provitamin A activity ß-carotene has 100 % provitamin A activity (Zeb and Mehmood 2004; Vílchez et al 2011) 32.3.5.1 Molecular Photovoltaic Nanomaterial Precursors The total energy, dipole moment, isotropic polarizability and molecular structure of the carotenoids make them eligible candidates for applications in dye-sensitized solar cells (DSSC) There has been a comparative study on the ionization potential and electron affinity of the carotenoids to validate them for the above purpose (Ruiz-Anchondo et al 2010) 32 Microalgal Rainbow Colours for Nutraceutical and Pharmaceutical Applications 32.3.5.2 Food Industry/Food Colourants Carotenoids are the precursors of various flavouring and odouring agents They also function as colour enhancers and hence have a wide use in the food and feed industry ß-carotene can be of use in food and beverages such as fruit juices, soft drinks and confectionery to improve their appearance and also because of their antioxidant properties (Christaki et al 2013) The application of carotenoids in the food industry is limited due to their poor water solubility and low bioavailability which can be overcome through their encapsulation into nano-emulsions (Qian et al 2012) 32.4 Conclusions Microalgal pigment production is the most significant area of research in the field of blue biotechnology Classically, pigments have been produced synthetically, but a rising demand for natural pigments has promoted large-scale cultivation of microalgae for pigment production The enzymes and genes required for the regulation and control of biosynthesis of pigment production need to be investigated along with their applications to enhance their productivity The extraction process of the pigments can be improved by simultaneous extraction of lipids or other bioactive molecules to offset the single product production cost Further to this, the interaction of pigments with other biological molecules and pigment-based nanostructure is an area which is still unravelled and can be explored in more detail Acknowledgements CP, TG and RM wish to thank AcSIR for Ph.D enrolment and CSIR for 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