南非多肉植物 Anacampseros rufescens 在逆境下葉部花青素與葉綠素濃度與光合作用生理的關係

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(1)國立臺灣師範大學生命科學系碩士論文. 南非多肉植物 Anacampseros rufescens 在逆境下葉部花青素與葉綠素濃度與光合作用生理的關係 Possible Ecophysiological Role of Anthocyanin and Chlorophyll Concentrations in Leaves of the South African Succulent Anacampseros rufescens (Portulacaceae) under Stress. 研究生：李宜蓁 Yi-Chen Lee 指導教授：林登秋博士 Dr. Teng-Chiu Lin Dr. Craig E. Martin 中華民國 102 年 7 月.

(2) Acknowledgement. I am very grateful to many people that have contributed in direct or indirect ways to this thesis. First of foremost, I would like to acknowledge the support of my advisor, Dr. Teng-Chiu Lin, for patient guidance, generous assistance and to give me opportunity to visit The University of Kansas. Without his support, I could not complete this thesis. Second, I am grateful for my co-advisor, Dr. Craig E. Martin, for guidance me designed and completed this project. The most important, he taught me the proper attitude about doing research. Third, I would also like to thank the comments and suggestions from committee, Dr. Yau-Lun Kuo. Many thank are also owed to lab colleagues and friends for their assistance and patience. Finally, I would like to thank my parents for their emotional support.. 2.

(3) Table of Content Acknowledgement ................................................................................................ 2 Table of Content .................................................................................................... 3 摘要 ............................................................................................................................. 5 Abstract ..................................................................................................................... 6 Introduction ............................................................................................................ 8 Anthocyanin ............................................................................................................ 8 The functions of anthocyanins ............................................................................ 9 Photoprotection .................................................................................................... 11 1. Protection of the photosynthetic apparatus .......................................................12 2. Protection from ultraviolet radiation ...................................................................13 Drought resistance ............................................................................................... 14 Objectives/Questions ........................................................................................... 15. Materials and Methods .................................................................................... 17 Plant Materials ..................................................................................................... 17 Chlorophyll fluorescence.................................................................................... 18 Chlorophyll concentration ................................................................................. 19 Anthocyanin concentration ............................................................................... 20 Statistical analysis ................................................................................................ 20. Results ...................................................................................................................... 22 Pigment concentration ........................................................................................ 22. 3.

(4) Correlation of chlorophyll fluorescence parameters with anthocyanin concentration (both light treatments combined) .......................................... 26 Correlation of chlorophyll fluorescence parameters with anthocyanin concentrations (separate light treatments) .................................................... 28 Drought treatment ............................................................................................... 31. Discussion ............................................................................................................... 38 Pigment concentration ........................................................................................ 38 Light treatment-chlorophyll fluorescence correlation................................. 40 Drought treatment ............................................................................................... 41. Conclusions ............................................................................................................ 44 Reference ................................................................................................................ 45. 4.

(5) 摘要. 花青素是一種水溶性黃酮類色素，廣泛分布在許多的植物組織而表現出紅或紫等顏色。花青素曾被認為是不具功能的次級代謝物，然而近年來的研究顯示，花青素可以影響植物反應及調適環境逆境的能力。本研究檢測葉花青素濃度與南非沙漠多肉植物 Anacampseros rufescens (Harv.) Sweet 調適逆境的關係。實驗處理為讓植物生長在高低光及有無缺水環境，透過實驗探討以下三個問題： 1、不同光度如何影響 A. rufescens 上下表皮花青素的濃度。2、花青素在光保護作用中所扮演的角色。3、缺水如何影響 A. rufescens 葉中花青素的濃度與其光保護作用的潛力。研究結果顯示，生長在高光環境中的植物，花青素濃度與葉綠素螢光值有顯著正相關，但在低光環境則無顯著相關性。缺水處理後，高低光生長的植物花青素及葉綠素濃度無顯著改變。然而在低光生長的植物，最大光化學效率（Fv/Fm）下降，惟下降幅度微小。總結來說，在高光環境生長的植物其花青素可能有光保護作用，本研究結果無法支持一些研究所指花青素在植物耐缺水所扮演的作用。本實驗結果有助於釐清沙漠多肉植物葉中的花青素所扮演的角色，且有助於了解其在南非沙漠的生物學與生態學特性。. 關鍵字：花青素、缺水、光保護作用、葉綠素螢光. 5.

(6) Abstract. Anthocyanins are water-soluble flavonoid pigments, often having a red or purple color, that occurs in many plant tissues. Although this pigment was originally considered a metabolic waste product, contemporary research has indicated that anthocyanins can significantly influence plant responses and adaptations to environmental stress. This study examined the relationship between leaf anthocyanin concentration and plant responses to environmental stress in the South African desert succulent Anacampseros rufescens (Harv.) Sweet. Plants were grown at high and low light levels under watered and drought treatments. Questions addressed by this study include: 1. How does light level during growth affect the anthocyanin and chlorophyll concentrations of the abaxial and adaxial halves of the leaves of A. rufescens? 2. What is the role of anthocyanin in photoprotection in the leaves of A. rufescens? 3. How does drought stress affect the anthocyanin and chlorophyll concentrations and potential photoprotection in the leaves A. rufescens? Anthocyanin concentrations positively correlated with light-adapted chlorophyll fluorescence in plants grown under high light. This, however, was not true for plants grown under low light. In the drought treatment, anthocyanin and chlorophyll concentrations in the high light and low light -treated plants were not affected by the drought treatment. In the low light treatment, however, Fv/Fm declined after drought, although the decline was small.. 6.

(7) In summary, the results of this study indicate that anthocyanin likely has a photoprotective function for plants under a high light environment. A specific role of anthocyanin in drought tolerance as reported by several studies was not supported by this study. The results of this study help to clarify a photoprotective role of anthocyanin in leaves of a desert succulent and provide ecophysiological insight into the ecology and biology of this South African desert species.. Keywords: anthocyanin, drought, photoprotection, chlorophyll concentration. 7.

(8) Introduction. Plants leaves include many organic pigments that occur in various colors, including chlorophyll, carotenoids, betalain and flavonoids. Flavonoids have a variety of functions, including UV protection, attraction of insect pollinators, plant growth regulation, etc. One of the most colorful flavonoids is anthocyanin. Besides coloration of flowers, anthocyanin has diverse functions in leaves. The function of anthocyanin on drought resistance and potoprotection are examined here.. Anthocyanin. Anthocyanins comprise a type of flavonoid that is modified by a glycoside (Harborne and Williams 2001). They are water-soluble pigments typically found in the vacuole of epidermal cells. The color of anthocyanins could be appearing red, purple or blue depending on the vacuolar pH (Neill and Gould 2000). They can be found in all plant tissues, including leaves, stems, roots, flowers, and fruits. A well-known function of anthocyanins, as well as other flavonoids is fruit and flower coloration, thus serving to attract insects that help in pollination and seed dispersal (Harborne 1965). Besides their function in reproduction, anthocyanins have multiple functions in leaves, which often appear red as a result. 8.

(9) Plants with red leaves are distributed throughout the plant kingdom from mosses to angiosperms (Lee 2002) and are found in growth habitats from polar regions to tropical rainforests and from deserts to freshwater lakes. Red leaves can be found in the uppermost tree canopy or in the forest understory (Gould 2004). Leaves of some species are red due to anthocyanins for their entire existence (Gould et al. 2000), whereas others are red for only one season in a year (Feild et al. 2001). Furthermore, certain stresses, especially drought, can stimulate the production of anthocyanins in leaves (Chalker-Scott 1999).. The functions of anthocyanins. When first discovered, plant scientists considered anthocyanins to be “secondary compounds” (Gould 2004), meaning they lack a direct function and are most likely a by-product of reactions with more immediate and direct functions. Now, however, several lines of research have clarified a diverse set of functions for this class of plant pigments (Gould 2004). Anthocyanins can influence how leaves respond to environment stresses, and they can constitute a critical pigment for plant survival under stress (Gould 2004). The multiple functions of anthocyanins include the following: 1. Photoprotection: anthocyanins can decrease photoinhibition and photobleaching when leaves are exposed to high light (Hamilton and Brown 2001). Because this function is the primary focus of the current 9.

(10) study, it is reviewed in greater detail below. 2. Cold hardiness: high concentrations of anthocyanins in plant tissues can improve tolerance to low temperature during the winter (Leng et al. 1993). 3. Drought resistance: when plants experience drought, anthocyanin concentrations may increase in leaves of xerophytes, reducing the osmotic potential of cells in the leaves (Hsiao 1973). Moreover, this osmotic effect can maintain cell turgor and, thus, leaf shape and orientation (Chalker-Scott 1999). 4. Antioxidative abilities: the results of a number of studies indicate that anthocyanins can play an antioxidative role in photosynthetic tissues, thus minimizing damage due to the production of oxidizing agents, such as free radicals (Hipskind et al. 1996, Manetas 2006, Osório et al. 2013). 5. Antiherbivory capabilities: many animals avoid eating red leaves because of an aposematic color (Gould 2004). Thus, anthocyanin may even have an aposematic function, mimicking the red color of resource-poor leaf litter (Furuta 1986; Lee 2001). 6. Protection of photolabile defense compounds: anthocyanins can protect herbivore- and pathogen-defense compounds that are easily degraded by light (Page and Towers 2002, Gould 2004).. In the review of environmental significance of anthocyanin Chalker-Scott 10.

(11) (1999) indicated that anthocyanins can help to resist multiple stresses through modification of compounds in vacuole to attenuate radiation and adjust osmotic pressure. The following are some important characteristics that contribute to diverse functions of anthocyanins. Because anthocyanins are water-solute pigments their accumulation in vacuole helps to adjust change osmotic pressure (Harborne and Williams 2001). Osmotic pressure can be changed by multiple environmental factors, including radiation, extreme temperature and water gradient etc. Therefore, the concentration of anthocyaninss in vacuole is closely related to plant’s ability to tolerate stresses (Gould 2004). Anthocyanins are glycosylated, so they could transport or bind sensitive monosaccharides during stressful environmental conditions (Chalker-Scott 1999). For example, the binding or transport of monosaccharides contributes to the hardening during cold period (Chalker-Scott 1999). In addition, anthocyanins acylated with hydroxycinnamic can attenuate or reduce UVB (Giusti et al. 1999).. Photoprotection. The most well-known and studied function of anthocyanins is photoprotection (Steyn et al. 2002), which is defined here as any of several mechanisms that can decrease damage from excessive light. Some well- known 11.

(12) non-photosynthetic, photoprotective pigments, e.g., anthocyanin, betalain, rhodoxanthin (Solovchenko 2010), and zeaxanthin, can absorb excitation energy from over-excited chlorophyll that might otherwise result in damage to the photosynthetic tissue (Demmig-Adams and Adams 1992).. 1. Protection of the photosynthetic apparatus. When leaves absorb excessive energy, plants may produce toxic compounds (e.g., free radicals) or degrade carbon fixation compounds, both of which reduce the photosynthetic efficiency of the leaves, a process referred to as photoinhibition (Long et al. 1994). During exposure to high light, anthocyanin in the epidermal cells may act as a light filter or “sunscreen” that can reduce over-excitation of the chlorophylls, thereby reducing radical production in the photosystems (Neill and Gould 2003). Some studies observed expanding leaves had high anthocyanin accumulation. Developing leaves are not photostable because their light utilization and abilities to dissipate extra energy are low (Close and Beadle 2003) and the accumulation of anthocyanin may help to increase photostability (Drumm-Herrel and Mohr 1982). Field et al. (2001) used red leaves and yellow leaves of the temperate tree redosier dogwood (Cornus stolonifera) to exam their photosynthetic responses to excess light. Photosynthetic efficiency of red leaves was inhibited by 60%, while that of yellow leaves was inhibited by 100%. The authors postulated that protection of the chlorophyll by anthocyanins is an important consequence of 12.

(13) leaf color change seen in temperate deciduous trees in the fall. However, not all studies provided consistent results of the role of anthocyanins on photo-protection. For example, Burger and Edwards (1996) found that photoinhibition levels were not significantly different between red and green leaves of coleus under high irradiances. In addition, Krol et al. (1995) found that photoinhibition levels were not significantly different in jack pine leaves (needles) with and without high levels of anthocyanin after exposure to high levels of white light. In these studies, it appears that anthocyanin did not protect the chlorophyll from photoinhibition in high radiation exposure (see Steyn et al. 2002). More studies are required before the role of anthocyanis on photo-protection can be clearly defined.. 2. Protection from ultraviolet radiation. The results of many studies indicate that flavonoids, including anthocyanins, can protect leaves from potentially damaging UV radiation (Woodall and Stewart 1998, Smillie and Hetherington 1999). Anthocyanins in particular have an acylated group which can absorb UV radiation (Alexieva et al. 2001) and, as a result, reduce damage to the plant (Takahashi et al. 1991, Caldwell et al. 1994, Stapleton and Walbot 1994). When the gene that is associated with the anthocyanin synthetic pathway was knocked down in Arabidopsis, leaves become highly sensitive to UV radiation damage (Li et al. 1993). Anthocyanins appear to attenuate UV radiation in a manner similar to that found for 13.

(14) phytochrome (Bowler et al. 1994). As is the case with anthocyanin, most of the colorless flavonoids are located in the leaf epidermal cells, thus preventing UV radiation penetration into the photosynthetic tissue. Unlike anthocyanin, the presence of colorless flavonoids have no influence on photosynthesis (DeLucia et al. 1992). Anthocyanin is also often distributed in palisade tissue, where it would absorb photosynthetically useful (or damaging) radiation. Therefore, the presence of anthocyanin in leaves often reduces photosynthetic efficiency (Burger and Edwards 1996, Gould et al. 2000).. Drought resistance. Drought can cause leaf dehydration and cell plasmolysis (Curtis et al. 1996), and water stress may lead to photoinhibition (Björkman and Powles 1984) and as such changes in chlorophyll fluorescence (Efeoğlu et al. 2009, Sperdouli and Moustakas 2012, Osório et al. 2013). Some suggest that drought could induce multiple responses and affect many metabolic processes in plants (Hsiao 1973). Anthocyanins have been suggested to play a drought-resistance role (Chalker-Scott 1999). For example, purple plants with anthocyanin appeared more tolerant to water stress than green plants (Bahler et al. 1991, Sherwin and Farrant 1998). However, drought did not always induce anthocyanin accumulation. Efeoğlu et al. (2009) measured anthocyanin concentrations by 14.

(15) three species of maize and found that anthocyanin accumulation was observed only in two species under drought stress. In fact, currently the possible function anthocyanin in drought stress is not well-understood. One of the objectives of current study is to examine how does drought stress affect the anthocyanin concentrations and potential photoprotection in the leaves A. rufescens?. Objectives/Questions. Although a number of studies provide evidences indicating that anthocyanin is an important pigment for photoprotection in photosynthetic tissues, there are also studies that provide contrary evidences for this function of this widely distributed pigment in the plant kingdom. Furthermore, fewer studies have examined the role of anthocyanin in protecting a plant from multiple stresses simultaneously. The South African succulent Anacamperos rufescens (Portulacaceae) was chosen for this study because the succulent leaves of this plant produce large amounts of anthocyanin, and the plant naturally occurs in highly exposed sites in the arid Little Karoo desert region of South Africa. This species has the abilitiy to tolerate the drought and high light exposure environments. Furthermore, the tissue distribution of anthocyanin in the leaves of this species is particularly interesting; high concentrations of anthocyanins typically occur in both the adaxial and abaxial epidermic of this plant. It was suggested that anthocyanins in adaxial halve leaves can absorb red 15.

(16) light to protect the chlorophyll (Hughes et al. 2008). However, the function of anthocyanins in abaxial halve leaves is still unknown.. Therefore, the primary questions addressed in this study include the following: 1) How does light level during growth affect the anthocyanin concentration of the abaxial and adaxial halves of the leaves of A. rufescens? 2) What is the role of anthocyanin in photoprotection in the leaves of A. rufescens? 3) How does drought stress affect the anthocyanin concentrations and potential photoprotection in the leaves A. rufescens?. 16.

(17) Materials and Methods. Plant Materials. Whole plants of Anacampseros rufescens (Haw.) Sweet (Portulacaceae) were collected in 2002 from shallow soil in depressions and cracks on light grey boulders (1-5m diameter) from undeveloped areas on personal property near Worcester (33° 38’ 42” S, 19°26’37” E) in the Little Karoo region of the Western Cape Province, transported to the University of Kansas, where they were planted in standard greenhouse soil in one-liter plastic pots and grown in a greenhouse until use. Environmental conditions in the greenhouse were under a natural photoperiod with a photosynthetic photon flux (PPF) ranging from 200μmol m -2 s -1 during cloudy conditions to 1000 μmol m -2 s -1 in full sunlight, relative humidity 50/80 % day/night and averaged air temperature ranges of 25-30 °C/15-20 °C day/night. Plants were watered at least once every 2d and fertilized weekly with 20 : 10 : 20 N : P : K (with traces of Mg, B, Cu, Fe, Mn, Mo and Zn) (Scott’s Peter Professional Water Soluble Fertilizer, Marysville, OH. USA). After growth in the greenhouse, 12 plants were moved to a growth chamber from October 2008 to August 2012; six plants in high light (HL) conditions, and six in low light (LL) conditions. Environmental conditions in the growth chamber were: 12-hour photo- and thermoperiod with PPFD of 130-260 μmol 17.

(18) m -2 s -1(HL) and 27-95 μmol m -2 s -1 (LL), leaf temperatures of 34 °C (HL) and 28 °C (LL), and air vapor pressure deficit (vpd) of 2.97 kPa (day)/0.24 kPa (night). PPFD was measured with a LI-COR (Lincoln, NE) LI-190SB Quantum Sensor and a LI-185B Light Meter. Leaf temperatures were averages of five leaves per plant and were measured with a Fisher (St. Louis, MO, USA) Model 4315 Infrared Thermometer. Plants were watered two times per week and fertilized every two to three months (see above for fertilizer details). Fluorescence and pigment concentrations were measured at the beginning of the experiment and after five, ten, and 200-205 weeks of treatment. In addition to the light treatment, a drought treatment was conducted at the end of the measurement period. All plants were drought-stressed by withholding water for 28 days. All the fluorescence and pigment concentrations were measured both before and after the drought treatment.. Chlorophyll fluorescence. Chlorophyll fluorescence was measured on leaves detached from plants (one leaf per plant) in the laboratory outside the growth chamber at 07:00am in the dark and again, using a different leaf, in the light at 12:30pm using a LICOR (Lincoln, NE) LI-6400 Portable Photosynthesis System with an LI-640040 Leaf Chamber PAM Fluorometer. Only the uppermost leaves having a width greater than 2 cm (chamber aperture diameter 2 cm) were selected for 18.

(19) measurement. It was necessary to measure fluorescence on detached leaves because the leaves were too densely packed on the stems of the plants to allow access to the fluorometer chamber. During both dark and light measurements, fluorescence parameters were measured three times per leaf and only after stability of the fluorescence signal (i.e., when FlrCV% < 1.0). Environmental conditions inside the fluorometer chamber were 20°C (dark) or 30°C (light), PPFD of 250 μmol m -2 s -1 (HL) and100 μmol m -2 s -1 (LL), dark/light vpd as in the growth chamber, and 500 µl l-1 CO2 concentration. Calculations of all fluorescence data were performed by the LI-COR software accompanying the fluoescence unit (Demmig-Adams et al. 1996, Maxwell and Johnson 2000, Martin et al. 2010).. Chlorophyll concentration. Following the dark fluorescence measurement of a leaf, it was sliced horizontally into two halves. Each leaf half was then weighed, immersed in 10 ml spectrophotometric grade DMF (N,N-Dimethylformamide, ACS), then placed in a refrigerator (4°C) for five to seven days, after which, the discs appeared colorless. After decantation of the DMF solution, its absorbance was measured with a Thermo Spectronic (Rochester, NY) Genesys 10 Spectrophotometer at 603, 647 and 664nm. Chlorophyll concentrations were determined according to Moran (1982). The discs were dried at 70°C until no 19.

(20) further weight loss occurred.. Anthocyanin concentration. Following the light fluorescence measurement of a leaf, it was treated identically as described above for chlorophyll determination, except each leaf slice was immersed in 10 ml acidified methanol (1% hydrochloric acid) for anthocyanin extraction according to (Pietrini and Massacci 1998, Feild et al. 2001, and Gould et al. 2002). Anthocyanin concentrations were calculated and expressed as absorbance at 532 nm divided by the dry weight of the leaf slice.. Statistical analysis. Mean chlorophyll concentration, and anthocyanin concentration in high light and low light treatment were compared using a Student’s t-test. Mean chlorophyll concentration, and anthocyanin concentration in the adaxial and abaxial halves of leaves were compared using a paired t test. The relationship between anthocyanin concentration and fluorescence parameters (Fv/Fm, Fv’/Fm’, qP, qN) was examined with Pearson’s coefficient correlation. Mean chlorophyll concentration, anthocyanin concentration, and fluorescence parameters (Fv/Fm, Fv’/Fm’, qP, qN) before and after drought were compared 20.

(21) using a paired t test with JMP 6 (SAS Institute, Cary, NC, USA). Means were considered to be significantly different when p ≤ 0.05.. 21.

(22) Results. Pigment concentration Chlorophyll concentrations of the adaxial halves of the leaves were greater than those of the abaxial halves of leaves in the low light treatment in wellwatered plants (p = 0.0218, Table 1), after the drought treatment (p = 0.0001, Table 1) and when all measurements were pooled (p < 0.0001, Table 1). Chlorophyll concentrations of the two leaf halves were not significantly different for plants grown in the high light treatment, regardless of water status (Table 1). Anthocyanin concentrations of the adaxial and abaxial leaf halves were not significantly different, regardless of light or water treatment (Table 1). The above results emphasize comparison of pigment concentrations of leaf halves of plants growing at two light levels. When comparing the data for the light levels, chlorophyll concentrations of the adaxial halves of the leaves and of whole leaves in the low light treatment were higher than those in the high light treatment but the difference was only significant after drought treatment (p = 0.006 for adaxial halves of the leaves and p = 0.050 for mean of the leaves, Table 2). For both halves (separately and averaged), anthocyanin concentrations were not significantly different between the high light and low light treatments both before and after the drought treatment (Table 2).. 22.

(23) Table 1. Mean (± SE) chlorophyll and anthocyanin concentrations of the adaxial and abaxial halves of leaves of Anacampseros rufescens under wellwatered and drought-stress conditions for plants grown under two light levels. The bold numbers indicate that p-values were smaller than 0.05. Well-watered. After drought. All data. N=35. N=23. N=58. Adaxial. 6.61±1.54. 4.47±0.82. 5.75±0.99. Abaxial. 6.34±1.26. 4.22±0.77. 5.49±0.83. 0.8351. 0.2638. 0.7343. Adaxial. 7.71±0.79. 7.26±0.33. 7.53±0.49. Abaxial. 6.83±0.85. 5.06±0.38. 6.14±0.56. 0.0218. 0.0001. <0.0001. Adaxial. 10.73±1.93. 4.98±0.91. 8.43±1.31. Abaxial. 9.78±1.55. 6.25±0.71. 8.37±1.01. 0.6147. 0.1624. 0.9562. Adaxial. 10.72±1.42. 7.96±1.55. 9.64±1.07. Abaxial. 7.80±1.36. 6.49±1.05. 7.28±0.92. 0.1399. 0.1664. 0.0597. Chlorophyll (mg/g) HL p LL p Anthocyanin (Abs/g) HL p LL p. 23.

(24) Table 2. Mean (± SE) chlorophyll and anthocyanin concentrations of plants in high light and low light treatments of Anacampseros rufescens under wellwatered and drought-stress conditions of the adaxial and abaxial halves of leaves. The bold numbers indicate that p-values were smaller than 0.05.. 24.

(25) Well-watered. After drought. All data. N=35. N=23. N=58. HL. 6.61±1.54. 4.47±0.82. 5.75±0.99. LL. 7.71±0.79. 7.26±0.33. 7.53±0.49. 0.5397. 0.0060. 0.1213. HL. 6.34±1.26. 4.22±0.77. 5.49±0.83. LL. 6.83±0.85. 5.06±0.38. 6.14±0.56. 0.7536. 0.3492. 0.5397. HL. 6.48±1.26. 4.34±0.79. 5.62±0.83. LL. 7.78±0.87. 6.16±0.31. 7.14±0.56. 0.4082. 0.0501. 0.1403. HL. 10.73±1.93. 4.98±0.91. 8.43±1.31. LL. 10.72±1.42. 7.96±1.55. 9.64±1.07. 0.9978. 0.1052. 0.4809. HL. 9.78±1.55. 6.25±0.71. 8.37±1.01. LL. 7.80±1.36. 6.49±1.05. 7.28±0.92. 0.3467. 0.8521. 0.4335. HL. 10.25±1.48. 5.61±0.70. 8.40±1.01. LL. 9.26±1.03. 7.22±1.23. 8.46±0.80. 0.590. 0.258. 0.962. Chlorophyll (mg/g) Adaxial P Abaxial P Whole leaves p Anthocyanin (Abs/g) Adaxial P Abaxial P Whole leaves p. 25.

(26) Correlation of chlorophyll fluorescence parameters with anthocyanin concentration (both light treatments combined). Combining data for both light treatments, and each light level separately anthocyanin concentrations did not correlate with maximum quantum efficiency of PSII photochemistry (Fv/Fm) (r = -0.03, p = 0.8156, Figure1A). In contrast, anthocyanin concentrations correlated positively with the efficiency of excitation energy capture by open PSII reaction center (Fv’/Fm’) (r = 0.42, p = 0.0011, Figure1B). Furthermore, anthocyanin concentrations correlated positively with the degree of photochemical quenching (qP) (r = 0.34, p = 0.0095, Figure1C) and of non-photochemical quenching (qN) (r = 0.41, p = 0.0015, Figure1D).. 26.

(27) Figure 1. Correlation between leaf anthocyanin concentrations and Fv/Fm (A), Fv’/Fm’ (B), qP (C), and qN (D) for all data from both light treatments at 5th, 10th, 200th, 202nd, 204th weeks of treatment (plants were drought-stress at 202ndand 204th week). All fluorescence parameters were measured on the same whole leaf of each of 12 plants. r and p were determined by Pearson’s correlation. ■, 5th week; ■, 10th week; ■, 200th week; ■, 202nd week; ■, 204th week. 27.

(28) Correlation of chlorophyll fluorescence parameters with anthocyanin concentrations (separate light treatments). In the high light treatment, anthocyanin concentrations did not correlate with Fv/Fm values (r = 0.01, p = 0.9380, Figure 2A). In contrast, anthocyanin concentrations correlated positively with Fv’/Fm’ values (r = 0.54, p = 0.0020, Figure 2B). Anthocyanin concentrations also correlated positively with qP (r = 0.47, p = 0.0090, Figure2C) and with qN (r = 0.51, p = 0.0038, Figure 2D). In the low light treatment, anthocyanin concentrations did not correlate with Fv/Fm (r = -0.03, p = 0.5153, Figure 3A), Fv’/Fm’ (r = 0.25, p = 0.2012, Figure 3B), qP (r = 0.13, p = 0.5053, Figure 3C), or qN (r = 0.27, p = 0.1697, Figure 3D) In summary, anthocyanin concentrations correlated positively with lightadapted fluorescence parameters only in the high light treatment, but not with dark-adapted fluorescence. Anthocyanin did not correlate with any fluorescence parameters in the low light treatment.. 28.

(29) Figure 2. Correlation between leaf anthocyanin concentrations and Fv/Fm (A), Fv’/Fm’ (B), qP (C), and qN (D) for plants in the high light treatment measured at 5th, 10th, 200th, 202nd, 204th weeks of treatment (plants were drought-stress at 202ndand 204th week). All fluorescence parameters were measured on the same leaf of each of 6 plants. r and p were determined by Pearson’s correlation. ■, 5th week; ■, 10th week; ■, 200th week; ■, 202nd week; ■, 204th week. 29.

(30) Figure 3.Correlation between leaf anthocyanin concentrations and Fv/Fm (A), Fv’/Fm’ (B), qP (C), and qN (D) for plants in the low light treatment measured at 5th, 10th, 200th, 202nd, 204th weeks of treatment (plants were drought-stress at 202ndand 204th week). All fluorescence parameters were measured on the same leaf of each of 6 plants) r and p were determined by Pearson’s correlation. ■, 5th week; ■, 10th week; ■, 200th week; ■, 202nd week; ■, 204th week. 30.

(31) Drought treatment. In both the high light and low light treatments, anthocyanin concentrations were not significantly difference before and after the drought treatment (p = 0.0561 for the high light treatment and p = 0.0518 for the low light treatment, Figure 4A). Likewise, chlorophyll concentrations were not significantly different before and after the drought treatment in both high and low light treatments (p = 0.8634; p = 0.0589, Figure 4B).. A. 31.

(32) 14. A. Anthocyanin (Abs/g). 12. High Light. Low Light. 10. 8. 6. 4. 2. 0 high light Before After. low light Before After. 10. B. 9. Chlorophyll (mg/g). 8. High Light. Low Light. 7 6 5 4 3 2 1 0 high light Before After. low light Before After. 32.

(33) Figure 4. Mean anthocyanin (A) and chlorophyll (B) concentration before and after drought treatment in both high light and low light treatment (lines projecting from bars are standard error; N=6). ＊ p < 0.05. 33.

(34) Fv/Fm values were not significantly different before and after the drought treatment for plants in the high light treatment (p = 0.2343, Figure 5A). In contrast, Fv/Fm values were lower after the drought treatment for plants in the low light treatment (p = 0.0260, Figure 5A). Fv’/Fm’ values were lower after the drought treatment in the high light treatment (p = 0.0010, Figure 5B), but these values didn’t change during drought in the low light treatment (p = 0.0689, Figure 5B). qP values were not significantly different before and after the drought treatment in the high light treatment (p = 0.0795, Figure 5C); however, such values were lower after the drought treatment in the low light treatment (p = 0.0023, Figure 5C). qN values were lower after the drought treatment in the high light treatment (p = 0.0013, Figure 5D), while no differences were observed before and after drought in the low light treatment (p = 0.0800, Figure 5D).. 34.

(35) 1. High Light. Low Light. A. *. 0.8. Fv/Fm. 0.6. 0.4. 0.2. 0 high light Before After. low light Before After. 1. B 0.8. High Light. Low Light. *. Fv'/Fm'. 0.6. 0.4. 0.2. 0 high light Before After. low light Before After. 35.

(36) 1. C 0.8. High Light. Low Light *. qP. 0.6. 0.4. 0.2. 0 high light Before After. low light Before After. D. 4. High Light. Low Light. 3. qN. * 2. 1. 0 high light Before After. low light Before After. 36.

(37) Figure 5. Mean fluorescence parameters, including Fv/Fm (A), Fv’/Fm’ (B), qP (C) and qN (D) before and after the drought treatment in both high light and low light treatment (likes projecting from bars are standard error; N=6). ＊ p < 0.05. 37.

(38) Discussion. Pigment concentration. Higher chlorophyll concentrations are typical for plants grown in low light environments (Bjorkman 1981, Boardman 1977). This was also the case for whole leaves of A. rufescens after 4 weeks of drought treatment. This result clearly reflected the large difference in chlorophyll concentrations of adaxial halves of leaves in plants after drought. In contrast to the above results, as well as many other studies, chlorophyll concentrations of leaves of plants grown under high and low light were not different. This was apparently the result of high variability in the data from well-watered plants; e.g., standard errors of the mean chlorophyll concentrations of plants under well-watered condition were 10% to 20% of the mean (see table 2). The primary reason for this light variability in the well-watered and drought data most likely resulted from the wide range of light treatment time for the well-watered plants (the 5th to 200th weeks) v.s. the drought-treated plants (the 202nd and 204th weeks) (Constable and Rawson 1980). Despite the sun/shade differences in leaf chlorophyll concentrations found in many plants (Bjorkman 1981, Boardman 1977), including A. rufescens under drought condition, chlorophyll concentrations of the abaxial halves of leaves were the same as (high light) or lower than (low light) those of adaxial surfaces 38.

(39) of these plants, regardless of watering condition. This unusual finding may be the result of the growth chamber environment in which the plants were grown. The inner walls of the chamber were bright white, which most likely reflected high irradiance to the abaxial surfaces of the leaves of the plants. Therefore, the abaxial surfaces of leaves intercepted more light than planned when the experiment was initiated. Anthocyanin concentrations in leaves of A. rufescens were not significantly different between plants grown at the two growth light levels, nor were they different between the adaxial and abaxial halves of the leaves of plants grown at either light level. These findings contrast with those of most past studies, in which more anthocyanin is typically found in photosynthetic tissues exposed to greater irradiances. There appear to be two possible reasons for this unusual result in this study of A. rufescens. First, as noted above, the growth environment of the plants resulted in exposure of the leaves, to relatively high irradiances on both leaf surfaces. Second, although not quantitatively examined, the leaves of plants of A. rufescens appear to synthesize some anthocyanins regardless of light levels (C.E. Martin, personal communication). One advantage of the latter is clear upon consideration of the natural environment of this species in arid region of South Africa. It is likely that both the adaxial and abaxial leaf surfaces are exposed to high irradiances as a result of the open habitats in which the plant grows, as well as the interception of high irradiances reflected off the rock on which the plants growth in the plant’s environment (see Material and Methods). 39.

(40) Light treatment-chlorophyll fluorescence correlation Fv/Fm, a measure of leaf’s efficiency in converting absorb light energy to photochemical activity (Bjorkman 1981) and a sensitive indicator of stress (Kooten and Snel 1990, Maxwell and Johnson 2000) was high (approximately 0.8) and did not differ significantly in A. rufescens, regardless of leaf halves (upper or lower), light level (high or low), or water treatment (well-watered and drought). This finding indicates that the plants in this study were not under high levels of stress. In addition, mean anthocyanin concentrations were not different among water treatment (well-water and drought) or leaf halves (abaxial and adaxial). In contrast, the amount of anthocyanin in whole leaves was positively correlated with Fv’/Fm’, which reflects the efficiency of utilization of light absorbed by the chlorophyll in the leaves. Thus, when leaf anthocyanin concentrations were low, the leaves did not efficiently utilize the light they absorbed by the chlorophyll. The qP and qN results indicate that this inefficiency was likely the result of temporary problems in the photochemical apparatus and a lack of thermal dissipation qN of the absorbed energy. The above finding applied to data combined for both light treatments. However, if the data from the high light and low light treatments were analyzed separately, anthocyanin concentrations correlated positively with fluorescence parameters only in the high light treatment. This finding is consistent with the results discussed above; plants in the high light treatment were more likely to 40.

(41) suffer problems such as photodamage. The lack of an apparent photoprotective role of anthocyanin in the low light-grown plants in this study may simply reflect the low probability of photodamage as a result of the low irradiances under which these plants were grown.. Drought treatment. Many studies have provided strong evidence that anthocyanin is an important pigment directly or indirectly attenuating drought stress (ChalkerScott 1999, Sperdouli and Moustakas 2012). In many species, drought stress induced or enhanced the accumulation of anthocyanin (Sherwin and Farrant 1998, Sperdouli and Moustakas 2012, Osório et al. 2013). In contrast, leaf anthocyanin concentrations were unaffected by drought stress in plants of A. rufescens. The results of this study imply that the photoprotective role of anthocyanin in this species is more important than a direct role in reducing the effects of drought stress. Fv/Fm values of plants in the low light treatment were significantly lower after the drought; however, the difference between values before and after drought was so small that the biological significance of this difference is questionable. On the other hand, a decrease in Fv/Fm values following drought stress is a common finding in many other plants (Efeoğlu et al. 2009, Sperdouli and Moustakas 2012, Osório et al. 2013). Although, Fv/Fm values in the high 41.

(42) light treatment did not significantly decrease after drought, the apparent decrease in Fv/Fm in the high light plants was similar in magnitude to the decrease in Fv/Fm observed in the low light plants. The light-adapted efficiency of light utilization (Fv’/Fm’) decreased after drought in the high light treatment, but did not significantly change in plants grown at low light. These findings indicate that the combination of drought stress and high light temporarily decreased the plants’ ability to efficiently utilize the light energy absorbed by the chlorophyll, but only in the high light treatment. Furthermore, nonphotochemical dissipation of absorbed light energy (qN) decreased with drought in the plants grown under high light, while photochemical activity (qP) did not change. Therefore, the decline in the efficiency of light energy utilization after its absorption by the chlorophyll can be ascribed, at least in part, to the loss of absorbed energy by nonphotochemical, thermal dissipation. Although the difference in Fv’/Fm’ before and after drought was not statistically significant in the low light treatment, qP decreased significantly, indicating that the rate of the light reactions (photochemical activity) declined with the drought treatment. The latter two results appear contradictory and indeed are, considering many past studies of stress effects on the manner in which absorbed light is utilized in the photochemical apparatus in plants under stress (Demmig-Adams and Adams 1992). Perhaps a greater sample size would result in similar declines in these two measures (Fv’/Fm’ and qP) in these plants grown under low light and drought-stressed. 42.

(43) It is clear that the manner in which light energy was utilized by the A. rufescens plants was different according to their growth light level. This may prove to be important to plants in situ as a result of microclimatic differences associated with the microhabitats in which these plants are found, e.g., shading by surrounding plants, stones, etc. vs. full exposure.. 43.

(44) Conclusions Leaf anthocyanin appears to play an important role in photoprotection in the South African succulent A. rufescens, which is likely important for survival under exposed conditions in an arid environment. For example, leaves with higher concentrations of anthocyanin exhibited less evidence of photoinhibition. Anthocyanin was found in both the adaxial and abaxial halves of the leaves, which should prevent damage due to high levels of irradiance from above the plants in exposed environments and from below the leaves due to reflection from rock surfaces in the plant environment. The photosynthetic (i.e., fluorescence) responses of plants under drought stress were variable and depended on the light level at which the plants were grown. Overall, anthocyanin in the leaves of this succulent from arid region in South Africa appears to be important for optimal ecophysiological function in a highly stressful habitat. This study is the first to explore the ecophysiological functions of anthocyanin in a South African succulent with a bifacial distribution of this pigment in the leaves of this xerophyte. It is also one of very few studies examining the potential importance of this photoprotective pigment on abaxial and adaxial leaf halves of a heliophyte. , I would like to thank the two advisors for tolerance my poor English.. 44.

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