The localisation, intracellular transport, and biosynthetic regulation of betalain plant pigments

<p>This thesis investigates the localisation, transport and biosynthetic control of betalain plant pigments to compare with the extensively researched anthocyanins. Anthocyanins and betalains appear similar, yet no plant naturally contains both pigment types. Due to this mutual exclusivity, betalain pigments are thought to functionally replace anthocyanins in many Caryophyllales. However, minimal research has been conducted to support this replacement hypothesis, resulting in limited knowledge of betalain pigment distribution and biosynthesis. The following series of experiments have added to this body of knowledge. Localisation of betalains was compared with that reported for anthocyanins. Histological analyses of 12 different betalain-producing species revealed similar pigment localisation to that of anthocyanic species. Similarities in pigment localisation suggest that these pigment types may have similar functional roles. The histological analyses also found that betacyanins and betaxanthins had differential localisation in several taxa. Organ- or tissue-specific distribution of betalain compounds suggests differing biological functions for betaxanthins and betacyanins. Hypotheses on betalain transport were tested using transgenic Arabidopsis thaliana lines capable of producing anthocyanins (PAP1-5), betalains (DOD-6), or both (DOD-6 x PAP1-5). Betaxanthins appeared to use vesicular transport, as betaxanthins were detected in small circular bodies within the cytoplasm. Furthermore, this observation suggests that betaxanthin formation occurred outside of the vacuole. DOD-6 was also crossed with Arabidopsis mutants, tt12 and tt19, which are deficient in proteins required for flavonoid vacuolar transport. Betaxanthin accumulation was reduced in both lines. In addition, DOD-6 was treated with transport inhibitors that affect anthocyanin accumulation. These experiments demonstrated that betaxanthins can utilise known flavonoid transport mechanisms, at least in this artificial pigment system. Regulation of betalain biosynthesis was analysed using Swiss chard (Beta vulgaris subsp. cicla cv. ‘Bright Lights’). Betalain production was induced through physical wounding of the lamina in red and white Swiss chard lines. Betalain pigments were produced around the wounding sites in the red line but not in the white line. Transcript level changes of betalain and flavonoid biosynthetic genes in these tissues were measured using real-time quantitative polymerase chain reaction analyses. Betalain biosynthetic genes were not up-regulated in the red line even though red pigments visibly accumulated. Rather, these genes were already expressed in the red line prior to wounding. Biosynthetic control of betalains may either be earlier in the pathway or at the post-transcriptional level. In contrast, all three flavonoid biosynthetic genes were up-regulated in response to wounding, indicating that expression of flavonoid and betalain biosynthetic genes are not co-regulated in Swiss chard. The final set of experiments examined the function of the two Beta vulgaris DOD genes (DODA and DODA1). Both genes were transiently expressed in nivea Antirrhinum majus dorsal petals and vacuum infiltrated with the betalain precursor L-DOPA. Expression of DODA1 but not DODA appeared to produce betalains. DODA-like genes have been found in anthocyanin-producing species, suggesting that this gene may not be involved in betalain biosynthesis. The overall findings from this research indicate that betalain pigment evolution of may have involved the utilization of anthocyanin transport machinery, but the regulatory control of the two pathways appears different.</p>

The localisation, intracellular transport, and biosynthetic regulation of betalain plant pigments

<p>This thesis investigates the localisation, transport and biosynthetic control of betalain plant pigments to compare with the extensively researched anthocyanins. Anthocyanins and betalains appear similar, yet no plant naturally contains both pigment types. Due to this mutual exclusivity, betalain pigments are thought to functionally replace anthocyanins in many Caryophyllales. However, minimal research has been conducted to support this replacement hypothesis, resulting in limited knowledge of betalain pigment distribution and biosynthesis. The following series of experiments have added to this body of knowledge. Localisation of betalains was compared with that reported for anthocyanins. Histological analyses of 12 different betalain-producing species revealed similar pigment localisation to that of anthocyanic species. Similarities in pigment localisation suggest that these pigment types may have similar functional roles. The histological analyses also found that betacyanins and betaxanthins had differential localisation in several taxa. Organ- or tissue-specific distribution of betalain compounds suggests differing biological functions for betaxanthins and betacyanins. Hypotheses on betalain transport were tested using transgenic Arabidopsis thaliana lines capable of producing anthocyanins (PAP1-5), betalains (DOD-6), or both (DOD-6 x PAP1-5). Betaxanthins appeared to use vesicular transport, as betaxanthins were detected in small circular bodies within the cytoplasm. Furthermore, this observation suggests that betaxanthin formation occurred outside of the vacuole. DOD-6 was also crossed with Arabidopsis mutants, tt12 and tt19, which are deficient in proteins required for flavonoid vacuolar transport. Betaxanthin accumulation was reduced in both lines. In addition, DOD-6 was treated with transport inhibitors that affect anthocyanin accumulation. These experiments demonstrated that betaxanthins can utilise known flavonoid transport mechanisms, at least in this artificial pigment system. Regulation of betalain biosynthesis was analysed using Swiss chard (Beta vulgaris subsp. cicla cv. ‘Bright Lights’). Betalain production was induced through physical wounding of the lamina in red and white Swiss chard lines. Betalain pigments were produced around the wounding sites in the red line but not in the white line. Transcript level changes of betalain and flavonoid biosynthetic genes in these tissues were measured using real-time quantitative polymerase chain reaction analyses. Betalain biosynthetic genes were not up-regulated in the red line even though red pigments visibly accumulated. Rather, these genes were already expressed in the red line prior to wounding. Biosynthetic control of betalains may either be earlier in the pathway or at the post-transcriptional level. In contrast, all three flavonoid biosynthetic genes were up-regulated in response to wounding, indicating that expression of flavonoid and betalain biosynthetic genes are not co-regulated in Swiss chard. The final set of experiments examined the function of the two Beta vulgaris DOD genes (DODA and DODA1). Both genes were transiently expressed in nivea Antirrhinum majus dorsal petals and vacuum infiltrated with the betalain precursor L-DOPA. Expression of DODA1 but not DODA appeared to produce betalains. DODA-like genes have been found in anthocyanin-producing species, suggesting that this gene may not be involved in betalain biosynthesis. The overall findings from this research indicate that betalain pigment evolution of may have involved the utilization of anthocyanin transport machinery, but the regulatory control of the two pathways appears different.</p>

Paper Chromatographic Separation of Plant Pigments a New Approach by using Akshya-Swagatika Solvent and Mobile Chromatogram Detection System (MCDS)

Chromatography is a term that refers to a group of laboratory techniques for separating mixtures. Chromatography works on the premise of solute partitioning between two phases or solvents. The technique of paper chromatography is commonly used to separate plant pigments based on their molecular weight. Plant pigments include chlorophyll-b, chlorophyll-a, carotenoid, and xanthophyll, which all have various molecular weights, colours, and absorption maxima. In this study, an attempt was made to see how a new solvent (Akshya-Swagatika solvent) can be used to separate plant pigments using paper chromatography, as well as a new detection method developed by us known as Mobile chromatogram detection system (MCDS) that can be used for compound identification and photographing. As shown in the table and photograph, pigments are separated by molecular weight and band colour, with carotenoid having the highest RF value and chlorophyll-b having the lowest. It was obvious that the Akshya-Swagatika solution could be used to separate plant pigments in paper chromatography. Photograph taken with a mobile chromatogram detection system that is much clearer than normal one. In developing countries, both solvent and detection systems are useful in explaining paper chromatography in a cost-effective manner. The MCDS detection system is cost-effective fast method which replaces traditional sophisticated detection procedures.

Assessment of the Suitability of Methods for Testing the Antioxidant Activity of Anti-Aging Creams

Anti-aging cosmetics are often sought after in order to slow down the aging process. Free radicals are one of the main causes of skin aging, and therefore antioxidants are used in anti-aging cosmetics. The aim of this study was to investigate which method is the most suitable for determining the antioxidant capacity of these products. Having samples extracted, the antioxidant capacity of the extracts obtained was determined by the following spectrophotometric methods: DPPH, Folin-Ciocalteu, FRAP, the ABTS method and the ferroion chelation method with ferrosine. The antioxidant capacity of the samples varied depending on the extract type and the method used. DPPH and ferroion chelation measurements with ferrosine were carried out in the part of the spectrum where plant pigments absorb. These pigments are often found in anti-aging products affecting these methods measurement results. The Folin-Ciocalteu method is suitable for researching the antioxidant capacity of hydrophilic extracts, but not lipophilic ones, where turbidity and the formation of a gelled ring occur. The FRAP method revealed similar results for all the samples and proved to be less sensitive than the others. The ABTS method for both types of extracts has proven to be the most suitable and sensitive method for determining the antioxidant capacity of anti-aging products.