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Astaxanthin Production in Tobacco Chloroplasts

METABOLIC ENGINEERING OF ASTAXANTHIN PRODUCTION IN TOBACCO CHLOROPLASTS
The Plant Journal 55, 857–868
Tomohisa Hasunuma, Shin-Ichi Miyazawa, Satomi Yoshimura, Yuki Shinzaki, Ken-Ichi Tomizawa, Kazutoshi Shindo, Seon-Kang Choi, Norihiko Misawa and Chikahiro Miyake.(2008).
Introduction
Astaxanthin (C40H52O4) is a carotein pigment and a fat soluable nutrient about 596.8 Dalton (Fig.1 and 2). The extended chain of conjugated (alternating double and single) double bonds is responsible for its red colour and antioxidant activity. Mainly used in medicnal purpose such as free radical scavanger and an immunomoderator and medical ingredient against degenerative diseases. They are also known as king of proteins. The natural astaxanthin is a powerful antioxident. This is responsible for red colouration in muscles of salmon, trout, lobster, shrimp and crab from their diet. Economically it is used as pigmentation agent in aquaculture feeds, food industries and cosmetics. This is mainly present in bacteria, fungi, algae, plants and animals. At this context of high demand with expensive market price, the high level production of natural astaxanthin is very important.

Figure 1. Structure of Astaxanthin Figure 2. Molecule of Astaxanthin
The production of astaxanthin by conventional gentic engineering of agrobacterium mediated gene transfer is less practicable and low yields. The majority of astazanthin produced synthetically and it contains some byproducts or intermediates. Sometimes conntaminated with other reaction of byproducts. So that its commerical application was restricted in aquaculture feed.
The metabolic engineering of tobacco plants used to bioproduction of astaxanthin (Fig.3). It is potential for large scale production by transgenic plants. The tobacco plant has the ability to accumulation of the high concentration of carotenoids in the thylakoid membrane and lipid globule in the plastid. The plastid engineering has the potential to reached high levels of protein accumulation in transformants. The two genes encoding Brevundimonas sp. (Marine bacterium) CrtW and CrtZ are introduced in the chloroplast by the translocation across the plastid membrane. The plastid vectors to insert the foreign genes between rbcL and accD through homologous recombination. The both genes were arranged as an operon under the control of a tobacco derived promoter Prrn.

Figure 3 Schematic representation of a potential carotenoid production pathway
In hybridisation to the rbcL, digestion of EcoRV OF crtZ/crtW integrated (Fig.4a). The integration of transgenes was verified by Southern blot techniques (Fig.4b). All transformants were accumulated the transgenes derived mRNA. An increase in dxs and psy transcript levels was observed in the ZW-2 and ZW-9 lines (Fig. 4 c and d).

Figure 4 Molecular characterization of transplastomic plants (a) Schematic representation of the plastid genome region of wild-type plants (b) Southern blot analysis of plastid transformants(c) Relative expression levels of crtW and crtZ in independent transgenic tobacco lines, (d) Relative expression levels of isoprenoid biosynthetic genes
Colour change was observed in transformed plant. No expression occurred in Wild–type plants (Fig.5). The nuclear transformants were removed the other accumulated unprocessed proteins not functional and interfere with the results. This is used to avoid the contamination.

Figure 5 Color changes in the aerial part (a) and flowers (b) of transplastomic tobacco
Research summary
The metabolic engineering to the production of desired compounds in plant tissues and to provide better understanding of genetically determined human metabolic disorders broadens the interest in this field (San et al., 1998) and elucidation of their control as determinants of metabolic function and cell physiology (Koffas et al., 1999). Carotenoids are synthesized within the plastids from the central isoprenoid pathway (Hirschberg, 1999). Metabolic engineering in plants is useful to increase the abundance of specific valuable metabolites (Uxue et al., 2013). Combinatorial nuclear transformation is a novel method for the rapid production of multiplex-transgenic plants (Zhu et al., 2008, 2009). Transformed DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, Lettuce plastid genome sequences insertion between the rbcL and accD genes and these are good for high level production of medicines, antibodies etc. (Kanamoto et al., 2006).
In the present study the growth and photosynthesis of tobacco transformants were analysed under light conditions of 300µmol photons m-2 s-1. There is no significant difference in the diameter of plants between the transplastomic and wild type plant at the flowering stage. The transplastomic tobacco was increased the production of astaxanthin by 2.1 fold and a novel variety of pigment carotenoid-4 ketoantheraxanthin was also produced.
Transplastomic tobacco that expressed two genes encoding CrtW and Crtz from Brevundimonas sp.SD212 accumulated large quanties of astaxanthin at concentration up to 5.44% of the total carotenoids and it showed the production of astaxanthin higher than the previous works.
Discussion
Astaxanthin is work as a super-powerful antioxidant and quickly eliminate free radicals and neutralize singlet oxygen (Capelli and Gerald 2013). In order to these economic values, so many studies were reported for the high level production of the astaxanthin from transgenic plants. Integration of Paracoccus sp.N81006 crtW and crtZ genes in to the tobacco nuclear genome accumulated trace amount of astaxanthin (Ralley et al., 2004), it is only one by tenth of those found in ZW-9 plants. A trace level of astaxanthin produced from transgenic potato leaf expressing the crtO gene of Synechosystis sp. PCC6803 under the CaMV 35S promotor (Gerjets and Sandmann, 2006). Complementation analysis by E.coli that revealed the CrtW (CrtO) and CrtZ (cyp175A) proteins respectivly Paracoccus sp.N81006 and synechosystis sp. PCC6803 (Choi et al., 2005, 2006, 2007). The higher levels of astaxanthin content in the transplastomic tobacco plants attributable to the Brevundimonas sp. SD212 CrtW and CrtZ. H. pluvialis bkt gene linked to the pea Rubsico small unit transit peptide sequence in carrot nuclear genome under the control of promoters (double CaMV 35S, Arabidopsis-ubiquitin or RolD from Agrobacterium rhizogenes and the transgenic carrot plant accumulate large amount of astaxanthin in leaves and roots (Jayaraj et al., 2007). But in the leaves the amount was only 0.0347 mg/g-1.
The cDNA of the gene CrtO from the alga H. pluvialis, encoding ?-carotene ketolase transferred to the plant under the regulation of the tomato Pds (phytoene desaturase) promoter. The transit peptide of PDS from tomato was used to target the CRTO polypeptide to the plastids (Mann et al., 2000). Chromoplasts in the nectary tissue of transgenic plants accumulated astaxanthin and other ketocarotenoids, changing the color of the nectary from yellow to red.
A transgenic Arabidopsis plants produced by ? carotene hydroxylase gene (Chyb) on oxycarotenoid biosynthesis that over-expressed Chyb under the control of a 35S promoter. Zeaxanthin and neoxanthin were two- to three-fold greater relative to the whole of violaxanthin, a final product in the xanlthophyll pathway, was 1.3-fold higher than the control and ? carotene declined as much as 2.4-fold and astaxanthin produced from transgenic plant (Cho et al., 2008).
Adonis aestivalis is synthesizing and accumulating large amounts of astaxanthin and other ketocarotenoids. An enzyme of ketocarotenoid biosynthesis in the flowers of Adonis aestivalis by the metabolic pathway with the help of E.coli is useful. The formation of astaxanthin requires only the addition of a carbonyl at the number 4 carbon of each beta-ring of zeaxanthin, a carotenoid typically present in the green tissues of higher plants (Cunningham and Gantt 2005). The production of astaxanthin from beta carotene by metabolic engineering pathway was also reported by Misawa 2009.
A major carotenoid in Sphingomonas sp. PB304, originally isolated from a soil was identified as astaxanthin dideoxyglycoside. Sphigomonas elodea-derived crtI and Nostoc sp. PCC 7120-dervied crtW genes were used for sequential analysis by sis enzymes pathway, including CrtX on the phytoene intermediate for the production by Tao et al., 2006.
Chlamydomonas reinhardtii has been genetically engineered with the ? carotene ketolase cDNA from Haematococcus pluvialis, bkt1 involved in the synthesis of astaxanthin and produced ketozeaxanthin (Leon et al., 2007). Methylomonas sp. strain 16a is an obligate methanotrophic bacterium used to produce, astaxanthin under the control of the native hps promoter in the chromosome. Canthaxanthin produced as main carotene and expression of two copies of crtZ and one copy of crtW led to the accumulation of a large amount of adonixanthin, more than 90% was astaxanthin.
Co-expression of the algal ?-carotene ketolase from Chlamydomonas reinhardtii and ?-carotene hydroxylase from Haematococcus pluvialis, a unique pair of enzymes identified to co-operate perfectly in converting ?-carotene to astaxanthin by functional complementation in E.coli. Expression of the two enzymes in tomato up-regulated most intrinsic carotenogenic genes and carbon flux into carotenoids resulted massive accumulations of mostly free astaxanthin in leaves (3.12 mg/g) and esterified astaxanthin in fruits (16.1 mg/g).This is a 16-fold increase of total carotenoid capacity and without affecting the normal growth and development of plant. The crop plant is useful to high yield production of astaxanthin for medicinal and other purposes (Huang 2013).
The result of the astaxanthin production from tobacco leaves compared to the result of the study of tomato leaves. As a result, the transplastomic tobacco yielded astaxanthin at 5.5 mg/g in leaves due to the plastid transformation is applicable only for a few model plants, making it difficult to copy the efficient pathway to plant tissues rich in chromoplasts and carotenoids for economical production of astaxanthin. But the amount of astaxanthin was high in transgenic tobacco (5.5mg/g) leaves than tomato leaves (3.12mg/g). Combined engineering of TCA and PPP modules had a synergistic effect on improving ?-carotene yield, engineering of MEP module resulted in 3.5-fold increase of ?-carotene yield (Zhao et al., 2013).
Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons were studied by Lu et al., 2013. Separation of foreign genes by polycistronic mRNA facilitates transgene stacking in operons studied by Zhou et al., 2007.These two studies are helpful to study the plastid transformation in plants.
Recently a similar works had done by Harada et al., 2014, the plastid genome of lettuce (Lactuca sativa L.) cv. Berkeley was site-specifically modified with the addition of three transgenes, which encoded (CrtZ) and CrtW) from a marine bacterium Brevundimonas sp. strain SD212, and isopentenyl diphosphate isomerase from a marine bacterium Paracoccus sp. strain N81106. Transplastomic lettuce plants were able to grow on soil at a growth rate similar to that of non-transformed lettuce cv. The germination ratio of the lettuce transformants (T0) (98.8 %) was higher than that of non-transformed lettuce (93.1 %). The transplastomic lettuce leaves produced the astaxanthin fatty acid (myristate or palmitate) diester (49.2 % of total carotenoids), astaxanthin monoester (18.2 %), and the free forms of astaxanthin (10.0 %) and the other ketocarotenoids (17.5 %). The artificial ketocarotenoids about 94.9 % of total carotenoids (230 ?g/g fresh weight). This is the first report of astaxanthin esters produced from a transgenic plant by transplastomic leaves. This study showed the high amount of astaxanthin esters (10.0%) from transplastomic lettuce plants.
The production of ketocarotenoids in plant tissues was a key objective of the METAPRO project. Plastid transformation was one approach. High astaxanthin is in tomato fruit. Fruit containing up to 25mg/g DW Astaxanthin in a mono-esterified form have been generated and the phenotype stable. The production of ketocarotenoids at very high level in tobacco, tomato (leaf and fruit) and potato (leaf and tuber) was reported by METRAPO. 2014.
Haematococcus pluvialis and Chlorella zofingiensis represent the most promising producers of natural astaxanthin. C.zofingiensis grown fast as phototrophically, heterotrophically and mixtrophically, high potential to be a better organism than H. pluvialis for mass astaxanthin production (Liu et al., 2014). H. pluvialis is the richest source of natural astaxanthin and is cultivated at industrial scale (Martin et al., 2003).
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Theories of Sexual Selection in Animals

It was more than 100 years ago that Charles Darwin formulated his ideas on sexual selection in The Descent of Man and Selection with regard to Sex (Darwin, 1871). He noticed that animals frequently possessed exaggerated traits which appeared detrimental to their survival, such as the large and decorative train of a peacock and bright plumage colors in many other birds. He recognized that such traits, despite being non-adaptive, may actually be beneficial if they conferred an advantage in terms of increased mating success to their bearers.
In sexually reproducing organisms, each of the offspring has one father and one mother, so the average reproductive success is equal for both males and females. However, if a male gains a disproportionate share of reproduction, he will take away reproductive opportunities from other males, leading to a high reproductive variance among males. On the contrary female will not take away reproductive opportunities from other females, leading to a smaller variance in reproductive success. The higher the reproductive variance, the stronger the effects of sexual selection.
However, theoretical analyses of sexual selection suggest at least three ways sexual selection might come about (1) Female choice: Female mate choice is the subject of a large area of research in behavioural ecology. Over the past three decades, many theoretical and empirical studies have investigated the patterns and consequences of female mating decisions (Andersson, 1994; Shuster and Wade, 2003; Barbosa and Magurran, 2006). If females choose from a variety of males and mate with those who will give those offspring having higher viability or fecundity, then alleles favouring such selection would be favoured and the system should persist. (2) Male-male sexual competition: if certain males are more able to prevent other males from having access to females, thereby sequestering this desirable resource for themselves, than those dominant males would be selected for and the system of sexual selection would persist. (3) Self-reinforcing choice: if there are alleles that, for whatever reason, predispose females toward males having an extreme character, then those males have an advantage. If that character is heritable, such that her male offspring also tend toward that extreme, then the character may be selected for. Fisher (1950) has termed this “runaway selection.” Alleles promoting this process may be selected even if they contribute nothing to, or possibly detract from, the viability of the males exhibiting them.
Despite the central role of female preference in sexual selection, and the importance of genetic variation in female preference, female preference is still poorly understood, prompting calls for investigations of the genetic variation in female preference (Heisler, 1984; Bakker and Pomiankowski, 1995; Wagner, 1998; Mead and Arnold, 2004).
Female mate choice has been demonstrated in numerous organisms, including invertebrates and vertebrates like frog, lizards, birds, mammals and has major consequences for the evolution of reproductive strategies (Andersson, 1994; Kokko et al., 2003). The success of males in achieving mating is often linked to the reproductive benefits which females derive (Jennions and Petrie, 1997; Bussiere et al., 2005). Males typically vary in their ability to provide benefits and determining how females detect differences among males in the benefits they offer has revealed much about the processes that drive the evolution of mate choice (Andersson, 1994).
Females use wide varieties of male traits such as larger morphological traits, bigger and brighter color patterns, more vigorous visual displays, and faster, longer, and louder calls to select their mates (Andersson, 1994; Ryan and Keddy-Hector, 1992). Female preferred males with such preferred traits to obtain can provide material resources that increase her or offspring fitness (direct benefits; Heywood, 1989; Price et al., 1993), alleles that increase offspring viability (good genes; Fisher, 1930; Grafen, 1990; Pomiankowski, 1988; Zahavi, 1975), or alleles that affect the attractiveness of male offspring (sexy sons; Fisher, 1930; Kirkpatrick, 1982).
To test whether females directly benefit from mating with preferred male or it is necessary to test the fitness consequences of variation in female mate preference. It is not important whether a study measures the fitness consequences of female preferences or measures the benefits obtained by mating female to account that variation among females is not confounded with variation among males. This is because females with stronger preferences may differ in a variety of ways from females with weaker preferences (Jennions and Petrie, 1997). However the fitness consequences of these differences can be confused with the fitness consequences of female mate preferences. Further, studies in animals have also shown that females which mated with more attractive male could produce more offspring, or invest more in each offspring they produce (Moller and Thorn hill, 1998). This observation suggests that females mating with preferred male obtain direct benefit if they provide nothing to avoid this problem by randomizing the association between trait attractiveness and direct benefit quality (Endler and Basolo, 1998).
If males vary in the direct benefits that they provide to females, or in the costs that they cause females to incur, and if females can directly or indirectly assess the benefits and/or costs of mating with males (i.e., direct benefit quality), then females’ preferences based on benefit quality should be favored. Whether preferences actually evolve will depend on a variety of factors, including the costs of being choosy and trade-offs between preferences and other traits.
The most difficult issues confronted in studies of female mate preference are why males should provide benefits to mated female. If male signals correlate with benefit quality, why male signals provides reliable information. To understand this problem by dividing it is potentially useful to direct benefits into three classes as follows. (a) Whether or not females can directly assess benefit quality of male prior to mating and (b) whether or not males use signals to attract females. These can be tested in species in which mating is resource independent because female choice occurs when males offer resources to females in the absence of signaling. (Thorn hill, 1976). If this is true, females can assess the mate for direct benefits prior to mating. In species where mating is a costly phenomenon because they have to face the risk of predation or a risk of parasite transmission, or if females only mate once per reproductive bout. In such situation selection may favor selective mating with those males that offer the highest-quality food items. Thus the female mate preference may favor the evolution of higher-quality direct benefits (e.g., Greater male investment in finding high quality food items). Such direct benefit of female mate preference that makes the evolution of female choice for direct benefits seem simple and straight forward.
Studies have also suggested pathways through which females mated with preferred male may obtain direct benefits. Females may obtain greater direct benefits by mating with the male showing some types of signals, independently of the costs and benefits (Reynolds and Gross, 1992). In animals in which mating is resource is independent in such species. The benefits that male animals provide to females includes nutrients, body parts, and secretions (Gwynne, 1982; Sakaluk, 1984; Thornhill, 1976); access to resources on a territory, including refuges, oviposition or nesting sites, and food (Howard, 1978) more sperm, more viable sperm, or better fertilization ability (Drnevich et al., 2001; Matthews et al., 1997); a variety of products transferred in seminal fluid, including nutritive and defensive compounds (Iyengar and Eisner, 1999; Markow, 1988); male protection, including protection from harassment by other males and from predators (Borgia, 1979) and male care of offspring, including care that frees females to engage in other activities and care that increases offspring fitness (Hill, 1991). Since these male contributions can affect female survivorship reproduction, offspring survivorship and reproduction. For two male contributions may refer to as direct benefits while later two male contribution may referred to as indirect benefits (Grether, 2010). Studies have also pointed out that females may also benefit from mating with certain males not because of the benefits they provide instead these males can impose lower costs on females. For example, females might risk damage from mating with certain male and she preferred to mate with males, which can lower the magnitude of damage (Grether, 2010)
There are growing list of evidence showing that in some taxes males display at fixed courtship, territories known as leks and these males provide only genes (i.e. Sperm) with their mates. (Non resource based mating systems) females receive no resources from males, yet females still show a preference in selecting their mate (Hoglund and Alatalo, 1995). This preference appears to be paradoxical because female of these species only receives genes from the male she selects (Kirkpatrick and Ryan, 1991; Tomkins et al., 2004). Therefore more studies are required to analyze the adaptive significance of age based female mate preference in insects.
The insect whose mating resource independent more appropriate to analyze age based female mate preference for both direct and indirect benefits. Therefore, studies involving accessory glands proteins and sperms are warranted.
Species of the genus Drosophila are one such genus whose mating is resource independently, where males does not show parental care or give a nuptial gift to the mated female. They have played an integral role in the development of sexual selection theory, and a great deal is known about the patterns and fitness consequences of female mate choice (Spieth, 1952; Partridge, 1980; Fowler and Partridge, 1989; Chapman et al., 1993; Gromko and Markow, 1993; Hegde and Krishna, 1997; Krishna and Hegde, 2003). Moreover, recent behavioural research revealed that male Drosophila varies greatly in its level of interest in females, providing evidence that males have also evolved to selectively mate (Gowaty et al., 2003). Furthermore, the reproductive biology of Drosophila is useful for investigating whether the female mate choice is influenced by male quality and the cost of choosing.
Initial studies involving D. melanogaster were concentrated on physiological changes associated with changes in parental age, molecular aspects, selection experiments and comparisons of populations that have been generated from individuals of different ages (Parsons, 1962; Wattiaux, 1968; Lints and Hoste, 1974; Ganetzky and Flanagan, 1978; Rose and Charlesworth, 1981; Partridge and Fowler, 1992; Roper et al., 1993; Chippindale et al., 1994; Orr and Sohal, 1994). However, they did not study the parental age effect on the direct benefits of female and offspring fitness.
In order to test good gene model requires the studies involving offspring fitness. However such experiments have been prohibited in many of the animals system. In spite of this very few attempts have been made in laboratory model organism i.e. in D. melanogaster (Price and Hansen, 1998). Recently, Prathibha and Krishna (2010) and Somashekar and Krishna (2011) have shown that females of D. ananassae and D. bipectinata prefer to mate with old aged males more frequently than middle or young aged males. Further, female mating with old aged males obtains both direct and indirect benefits. However, their studies did not involved accessory glands protein and sperm traits. Santhosh and Krishna (2013) involving accessory glands and sperm traits in D. bipectinata have shown that female mating with older males obtain a greater quantity of Acps and sperms than females mated with young or middle aged males. Further in D. melanogaster Abolhasan et al.,(2015) in their study also found that female mate with young males more frequently than middle aged and old males. They also showed that female mated with young male had obtained greater direct benefits (greater quantity of Acps and sperms) than those females mated with either middle aged or old males. Experimental evidences of these studies in species of Drosophila suggest that female mate preference for male age may be an indirect way of assuming the male ejaculate quantity. Therefore more studies of this type in the genus Drosophila is very much warranted to frame a hypothesis or generalization with regard to the female preference for male age to understand age based female preference in species of Drosophila. Until more species and genera are studied, it will be difficult to draw firm conclusions. Hence, more studies are needed in this regard.
Therefore present study has been undertaken in D. malerkotliana. It is a cosmopolitan species and belongs to a member of the bipectinata complex of the ananassae subgroup (Bock, 1971; Bock and Wheeler, 1972). It has a wide ecogeographical distribution ranging from India through South east Asia and New Guinea to Fiji and Samoa in the Pacific (Bock and Wheeler, 1972). It is a common occurrence in the Indian subcontinent and has attracted the attention of various Indian workers who are using this species for past few years. They have carried out extensive studies on population and behavior genetics of this species and have established the phylogenetic relationship between D. malerkotliana and other members of the bipetinata complex based on chromosome analysis, hybridization studies and isozyme analysis (Yang et al., 1972; Jha and Rehman, 1972; Hegde and Krishnamurthy, 1979; Singh et al., 1981; Hegde and Krishna, 1997). These findings provide interesting and important information concerning certain aspects of evolutionary genetics of this species. However, in this species it is not known whether females of this species discriminate males on the basis of their age classes, if so what its effect on female direct fitness benefits. Therefore present investigation has been undertaken in D. malerkotliana with the following objective.

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