Spatial and Temporal Variation of Carotenoids in Four Species of Trentepohlia (Trentepohliales, Chlorophyta)

to most of the tree barks, walls, rocks, and electric poles because of high accumulation of carotenoids in their filaments. This study emphasised the seasonal changes in carotenoid content amongst four different species of Trentepohlia , that is, T. diffracta , T. arborum , T. umbrina , and T. abietina , collected from four different substrata, that is, wall, rock, electric pole, and three types of tree bark (smooth, fissured, and rough). Quantitative estimation of different carotenoids, namely, 𝛽 -cryptoxanthin, lutein, and 𝛽 -carotene, from HPLC chromatogram peak showed a significant seasonal variation in all of the four species and 𝛽 -carotene was threefold more in winter compared to summer. Amongst the selected species, T. diffracta collected from wall contained the highest amount of 𝛽 -carotene both in summer and in winter, followed by T. arborum from rock and T. abietina from bark, and least amount was in T. umbrina collected from electric pole. Comparing the carotenoid content in Trentepohlia abietina growing in different types of barks, sample from smooth bark had the highest amount of carotenoids both in summer and in winter, followed by the sample from rough bark, and lowest amount was noted in fissured bark.


Introduction
Carotenoids are naturally occurring pigments synthesized by plants, algae, and photosynthetic bacteria. Carotenoids are classified into two classes, carotenes or carotenoid hydrocarbons, for example, lycopene, -carotene, and -carotene, and xanthophylls or oxygenated carotenoids, for example,cryptoxanthin, lutein, and zeaxanthin [1]. Carotenoids have received tremendous attention because of their various functions in plants, animals, and humans. In plants, carotenoids have the important antioxidant function of quenching singlet oxygen formed during photosynthesis [2]. These compounds are precursors of vitamin A and retinoid compounds required for morphogenesis in animals and humans [3,4]. Carotenoids are widely utilised in pharmaceutical purposes as health supplements and in food industries as natural colorants [5].
-Carotene and -cryptoxanthin are two carotenoids that show essential functions in humans where they serve as source of vitamin A [6,7]. Besides being precursor of vitamin A, -carotene and -cryptoxanthin are excellent antioxidants [8][9][10]. -Cryptoxanthin and lutein have got anticancer properties [11][12][13]. Moreover lutein is a good antioxidant as well [14,15] and it is one of the macular pigments that play an important role in maintaining eye health and in reduction of macular degeneration [16,17].
Trentepohlia Martius, belonging to the order Trentepohliales and class Ulvophyceae, is a dominant subaerial green alga of Shillong region and grows abundantly on different substrata, whereby its presence imparts yellow, orange, and red colours to the substrata due to accumulation of carotenoids in the filaments. Frequent occurrence of Trentepohlia on rocks, buildings, and tree barks had been reported by many workers [18][19][20][21][22][23]. Presence of specific carotenoids in Trentepohlia iolithus had been well documented by Kjosen et al. [24] and by Nybraaten and Liaaen-Jensen [25] whereas eight types of carotenoids had been reported in Trentepohlia gobii [26]. Abe et al. [27,28] had demonstrated the accumulation of carotenoids in T. aurea and the simultaneous production of  [33]. Hence the present study emphasised on studying the seasonal variation of carotenoids in some dominant species of Trentepohlia growing luxuriantly on different substrata with the aim of assessing the potential of some Trentepohlia species as source of some important natural carotenoids such as -carotene,cryptoxanthin, and lutein.

Sampling Area.
Shillong the capital of Meghalaya State which lies at an average altitude of 1496 m a.s.l. and between latitude 25 ∘ 30 N and longitude 91 ∘ 00 E was selected for sampling of algal species. Shillong experiences a subtropical highland climate, where summer is cool and rainy with an average total rainfall of more than 2100 mm annually, while winter is usually cold and dry. The four main seasons of Shillong are spring, monsoon or summer, autumn, and winter. Spring usually runs from March to April or May while monsoons arrive in June and rains almost until the end of August or sometimes continues right to the end of September. October and November are the transition months between rainy and winter seasons. The period between December and February is winter and is characterized by cold and dry weather. Collection of samples was done at several locations within Shillong area during summer (June, July, and August) and winter (December, January, and February) of 2014-2015. Details of total monthly rainfall and average monthly minimum and maximum temperature of Shillong area for each sampling month are given in Table 1.

Extraction.
The entire process of extraction, saponification, and spectrophotometer estimation of carotenoids was done following the procedure of Britton [39] with slight modifications. 1 gm of freeze-dried algal filaments was homogenized with a mortar and pestle in a dark room and extracted repeatedly with acetone in the presence of butylated hydroxyl toluene (BHT) as antioxidant. The homogenate was then centrifuged and the supernatant was collected. The solid residue was recovered for further extraction. The extraction procedure was repeated until extracting solvent was colourless. The combined extract was filtered through Whatman paper number 42 to remove the residue. The acetone extract was then mixed with equal volume of ethyl ether in a separating funnel and 3% sodium chloride solution was added until two layers separated. The upper combined ether extracts containing the carotenoids were washed three times with distilled water to remove the extracting solvent (acetone). For saponification, the above extract was dried completely and 40% of methanolic KOH was added; the mixture was then allowed to stand in the dark at room temperature for about 12 hours (overnight). Equal amount of ethyl ether was added followed by sodium chloride solution till the upper ethereal phase was separated. Then the upper part was washed with distilled water. This saponified extract was dried and finally dissolved in mobile phase and filtered through 0.45 m membrane filter paper before injection.

Separation and Quantification of Carotenoids.
The carotenoids were separated and identified following the procedure of Machmudah and Goto [40]. Carotenoids extracts were analysed using an HPLC Perkin Elmer series 200 equipped with Perkin Elmer Brownlee C18 column (5 m particle sizes, 150 × 4.6 mm). The mobile phase consisted of isocratic solvent of methanol and acetonitrile in the ratio 90 : 10 and flow rate was maintained at 1.0 mL/min. Carotenoids were monitored at 450 nm using UV-visible detector. The identification of various carotenoids in Trentepohlia was carried out by comparing the retention time and absorption spectra of unknown peaks with reference standards and estimation of the quantity of each carotenoid was carried out from the chromatogram peak areas.

Statistical Analysis.
All experiments were carried out with three replicates each. Two-way analysis of variance (ANOVA, < 0.05) was used to find out the significant difference in amount of total carotenoids and individual carotenoid between season (i.e., summer and winter) and amongst the different species of Trentepohlia (i.e., T. abietina, T. arborum, T. diffracta, and T. umbrina).

Results
Quantitative estimation of total carotenoids in all four species of Trentepohlia, that is, T. abietina, T. arborum, T. diffracta, and T. umbrina, by spectrophotometer showed a significant seasonal variation. Winter samples contained significantly higher total carotenoids content compared to that in summer samples. Among the four species, total carotenoids content was highest in Trentepohlia diffracta collected from cemented wall both in summer (38.96 mg g −1 ) and in winter (41.19 mg g −1 ). Total carotenoids content was low in T. umbrina collected from electric pole where it was only 36.06 mg g −1 in summer and 38.92 mg g −1 in winter ( Figure 1). Two-way analysis of variance (ANOVA) showed a significant difference in total carotenoids content between winter and summer ( = 0.011), but there was no significant difference amongst the different species of Trentepohlia ( = 0.678).
High Performance Liquid Chromatography (HPLC) chromatograms of different carotenoids in four species of Trentepohlia are given in Figure 2. The carotenoids separated by HPLC and identified by comparing with reference carotenoid standards were lutein (retention time 4 min),cryptoxanthin (retention time 7 min), and -carotene (retention time 10.3 min). Quantitative estimation of carotenoids from the peak area in all four species showed a similar trend where the quantitative amount of -cryptoxanthin, lutein, and -carotene was more in winter than in summer (Table 3). Among the three carotenoids studied, -carotene was severalfold higher compared to lutein and -cryptoxanthin. Remarkably the increase in level of -carotene in winter samples was almost threefold compared to that of summer. In     winter, the highest amount of -carotene was observed in T. diffracta collected from wall (974.34 ± 15.54 g g −1 ), followed by T. arborum collected from rock (605.71 ± 3.43 g g −1 ) and T. abietina collected from bark (585.27 ± 6.66 g g −1 ), and least amount was in T. umbrina collected from electric pole (520.17 ± 11.85 g g −1 ). Lutein and -carotene content showed significant seasonal variations, but when compared between four species of Trentepohlia collected from four substrata, no significant differences could be observed ( Table 4).
Assessment of carotenoids content in Trentepohlia abietina collected from different types of barks revealed that sample collected from smooth bark had maximum amount of carotenoids (lutein, -cryptoxanthin, and -carotene) both in summer and in winter, followed by sample from rough bark and minimum amount was noted in fissured bark of Pinus kesiya (Table 5). -Carotene in winter sample collected    Journal of Botany from smooth bark of Eucalyptus sp. was as high as 622.82 ± 1.10 g g −1 . Significant difference in quantity of -carotene was recorded in Trentepohlia abietina collected from different barks types ( = 0.008) while in case of Lutein andcryptoxanthin there was no significant difference (Table 6).

Discussion
A significant seasonal variation was clearly observed in this study where carotenoids were higher in winter compared to summer. High content of carotenoids in winter season with threefold increase in quantity of -carotene in all four species of Trentepohlia could be due to light, water, and temperature stresses. Shillong, lying at an average altitude of 1496 m a.s.l., experiences high light irradiance during daytime accompanied by dryness and low average temperature (2.3 ∘ C-18 ∘ C), which could be the reason for the enhancement of carotenoids production during winter. Increased production of carotenoids at high light intensity had been reported by Tan et al. [41] in Trentepohlia odorata and by Abe et al. [27] in Trentepohlia aurea. Low temperature along with high light intensity had been reported to induce carotenogenesis and enhanced -carotene production [42][43][44]. Ben-Amotz [45] reported that there was a fourfold increase in the 9-cis/alltrans -carotene ratio in Dunaliella bardawil when culture temperature was lowered from 30 ∘ C to 10 ∘ C. Furthermore, it was reported that disruption of cell membrane integrity takes place at low temperature and thereby making the algae more susceptible to photooxidation [46,47]. Hence, high amount of carotenoids is accumulated in some microalga as a protective measure to overcome photoinhibition and oxidative stress [48][49][50][51][52]. Similarly, these four species of Trentepohlia (T. abietina, T. arborum, T. diffracta, and T. umbrina) accumulated large quantity of carotenoids, especiallycarotene during winter, where temperature is low and photon irradiance is high accompanied by severe dryness as a strategy to protect the cells from photodamage or injury. On the other hand low amount of carotenoids content in summer samples of Trentepohlia could be attributed to high rainfall which is a common phenomenon in this region. This region is known as one of the wettest regions of the world. Taken as total of all the three summer months together (June, July, and August) 755.8 mm of rainfall was recorded during the experimental period. Constant supply of water or moisture in the form of heavy rainfall accompanied by cloudy days might have added to low carotenoids production in summer. According to Mukherjee et al. [31] low carotenoid content in T. aurea and T. cucullata in Assam during summer could be due to high temperature and cloudy days which was unfavourable for the growth of Trentepohlia. The highest accumulation of carotenoids in T. diffracta collected from wall and T. arborum collected from rock could be due to high light and water stress which consequently resulted in production of more carotenoids in the filaments for protection against oxidation, since the bare rocks and walls were more exposed to sunlight and dryness. Bartley and Scolni [53] documented that carotenoids played an important role as light-harvesting pigments and protect the photosynthetic apparatus from photooxidative damage under excess light conditions. Again the significant difference in amount of -carotene in Trentepohlia abietina collected from different bark characters showed that smooth bark played an important role in controlling the growth and carotenoids production in Trentepohlia. Low moisture holding capacity and full exposure to light could be the reasons for maximum accumulation of carotenoids to overcome those stresses in smooth bark.

Conclusion
This study had provided valuable information on some important carotenoids biosynthesized by Trentepohlia, whereby assessment of three important carotenoids content from four different species of Trentepohlia, that is, Trentepohlia abietina, T. arborum, T. diffracta, and T. umbrina, collected from different substrata had shown higher amount of -carotene accumulation which highlighted the potential of utilising Trentepohlia for the production of -carotene.