盐地碱蓬甜菜红素的鉴定及其生物合成的生理机制研究
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摘要
盐地碱蓬在滨海的潮间带或者部分涝洼积水地带在整个生长期植株地上部分皆为紫红色,而在地势较高或者距离海边较远的地方植株则呈现绿色。作为一种真盐生植物,盐地碱蓬的耐盐机理已得到了比较广泛的研究。但是,盐地碱蓬红色素为哪一类色素?这类色素的特性及生物学意义如何等至今尚未见报道。本课题主要是对盐地碱蓬红色素进行分离鉴定,并对影响红色素合成的主要环境因子以及红色素合成的生理机制进行深入研究。
研究结果表明:盐地碱篷红色素溶于水而不溶于纯有机溶剂、在酸性条件下稳定且为红色、在碱性条件下不稳定且为黄色、最大吸收峰为538 nm,根据这些特性我们初步确定该类红色素为甜菜红素(Betacyanins)。盐地碱篷甜菜红素在体外对高温、强光、碱和氧化剂敏感,而在低温、黑暗、酸性环境和没有氧化剂的条件下稳定。盐地碱篷甜菜红素体外清除超氧阴离子自由基的能力不如VC,而清除羟自由基的能力远高于VC。
光照抑制盐地碱蓬体内甜菜红素合成,黑暗、低温、高盐环境有利于盐地碱蓬甜菜红素积累,其中萌发期黑暗处理是诱导甜菜红素积累的最重要因子之一。盐地碱蓬种子暗培养3天得到的幼苗是紫红色,光照条件下培养3天得到的幼苗则是绿色。
用不同浓度的H_2O_2浇灌盐地碱蓬种子、喷洒5天苗龄盐地碱蓬幼苗的叶片、注射10天苗龄幼苗的叶片,测定其对盐地碱蓬甜菜红素含量的影响。结果显示不同方式的H_2O_2处理对盐地碱蓬不同器官的甜菜红素含量影响明显不同,H_2O_2浇灌后根中的H_2O_2含量增加最大,而叶片中甜菜红素含量增加最明显,但是叶片中的H_2O_2含量和根系中的甜菜红素含量增加却不显著。H_2O_2喷雾和注射虽然能明显提高叶片中的H_2O_2含量,但是对叶片甜菜红素含量却没有显著影响。器官中的H_2O_2含量和甜菜红素含量不成正比,叶片甜菜红素含量主要受到根系H_2O_2含量而不是叶片和茎H_2O_2含量的影响。因此,我们得出结论:H_2O_2浇灌引起的过氧化胁迫信号首先被根系感知,然后根系合成了某种可以传递到叶片并直接诱导甜菜红素合成的物质。
培养基中添加不同浓度的Ca~(2+)可以促进暗培养条件盐地碱蓬甜菜红素含量,钙螯合剂EGTA、离子通道抑制剂LaCl_3、CaM蛋白抑制剂CPZ和W7都可以抑制黑暗诱导的甜菜红素积累,但是这些处理对光培养条件下甜菜红素含量没有影响。黑暗和光照条件下盐地碱蓬甜菜红素含量都随内源钙的降低而降低。虽然CPZ和W7对光照条件CaM蛋白活性有抑制作用,但对光照条件下甜菜红素含量却没有明显影响。这些研究结果说明外源Ca~(2+)及CaM系统只参与了黑暗诱导的甜菜红素合成过程,内源钙同时参与了黑暗和光照条件下盐地碱蓬甜菜红素的合成过程。
在盐地碱蓬体内,酪氨酸酶活性只在黑暗条件下培养得到的明显积累甜菜红素的幼苗中检测到,而在光照条件下培养得到的幼苗中则检测不到,黑暗似乎是保持酪氨酸酶活性必需的条件。而且,当黑暗条件下培养得到的紫红色芽苗转入光照条件下后,酪氨酸酶的活性伴随着甜菜红素含量下降而逐渐降低。这说明酪氨酸酶参与了黑暗诱导的盐地碱蓬甜菜红素的合成。光照和黑暗调节盐地碱蓬甜菜红素合成和降解是通过调控酪氨酸酶活性实现的。
盐地碱蓬幼苗中CRY2蛋白在黑暗条件下积累,在蓝光条件下降解,这不仅和蓝光条件下甜菜红素含量下降的趋势一致,而且还和甜菜红素合成的关键酶酪氨酸酶的活性降低一致,这说明蓝光条件下CRY2蛋白在甜菜红素的降解过程中起着非常重要的作用,CRY2蛋白的降解是启动这种调控作用的关键。
As a halophyte, physiological and molecular responses of Suaeda salsa to salinity stress have been extensively studied. But nothing is known about the identification and the biosynthesis regulation of the red pigments in S. salsa. This paper focuses on the identification of the red pigments and the effects of abiotic stressors on their accumulation in S. salsa.
The results showed that these red pigments were insoluble in organic solvents but free in water, the pigments were red-violet and stable under acidic condition while yellow and unstable under alkaline condition, and they absorbed the highest value at wavelength near 538 nm. These results suggested that these red pigments in S. salsa are not anthocyanins but betacyanins.
The betacyanins extracted from S. salsa were sensitive to high temperature, high intensity of light, alkali and oxidant in vitro, and keep stable under low temperature, dark, acidic and free of oxidants conditions. The scavenging superoxide (O2-) ability of the betacyanins from S. salsa was lower than that of vitamin C, while the scavenging hydroxyl radical (OH·) ability of the betacyains was much higher than that of vitamin C in vitro.
Light suppressed betacyanin accumulation and enhanced their decomposition in vivo. Darkness, low temperature and high salinity enhanced betacyanin accumulation, and darkness at the germination phase is one of the most important environmental factors for the betacyanin accumulation in S. salsa.
Seeds, 5-d and 10-d old seedlings of C_3 halophyte S. salsa were watered, sprayed and infiltrated with 0, 0.10%, 0.33% and 1.00% H_2O_2 solution to examine whether H_2O_2 is involved in the betacyanin accumulation. H_2O_2 treatments led to the most significant betacyanin accumulation in the shoots of S. salsa seedlings when seeds were watered with H_2O_2 solution. The increase of betacyanin content was the highest and that of H_2O_2 content was the lowest in leaves, while the increase of betacyanin content was the lowest and that of H_2O_2 content was the highest in roots of seedlings obtained from seeds watered with H_2O_2 solution. When leaves of 5-d and 10-d old seedlings were sprayed and infiltrated with H_2O_2 solution, the H_2O_2 content was enhanced significantly, but the increase of betacyanin content was not notable in leaves, and the content of betacyanin and H_2O_2 was also at a lower level in roots. The H_2O_2 content in organs of S. salsa seedlings was not proportional to betacyanin content, and the betacyanin accumulation in leaves was correlated with the H_2O_2 production in roots rather than that in leaves and stems. These results suggested that the oxidative stress signal leading to betacyanin production in H_2O_2 treatments may be perceived by roots initially, then the signal was transferred to leaves; the signal transduction was performed and betacyanin accumulation was induced in leaves of S. salsa.
Seeds of S. salsa were cultured both in the dark and light for three days. Addition of Ca~(2+) in the 1/2 MS nutrient solution promoted betacyanin accumulation in the dark, and Ca~(2+ depletion by EGTA suppressed the darkness-induced betacyanin accumulation in shoots of S. salsa. Ca2+ channel blocker (LaCl_3) also inhibited the darkness-induced betacyanin accumulation. The highest activity of CaM and the maximum betacyanin content decreased by 51% and 45% in shoots of S. salsa seedlings treated with a potent CaM antagonist, chlorpromazine (CPZ), in the dark, respectively. Furthermore, another CaM antagonist, W7, also inhibited the activity of CaM and the dark-dependent betacyanin accumulation, whereas its less active structural analog, W5, had little effect on the responses to dark of S. salsa. These results suggest that Ca~(2+), Ca~(2+)-regulated ion channels and CaM play an important role in the darkness-induced betacyanin accumulation in shoots of C_3 halophyte S. salsa.
Seeds of the halophyte S. salsa were cultured in the dark for three days and betacyanin accumulation was induced significantly in cotyledons of S. salsa seedlings. Accumulated betacyanin in cotyledons of the dark-grown seedlings decomposed with time in light. As the betacyanin content declined, the hydroxylation and oxidation activity of tyrosinase extracted from cotyledons of the dark-grown seedlings decreased with time in light. The apparent molecular mass of tyrosinase in cotyledons of S. salsa seedlings was about 60 kDa estimated by SDS-PAGE, enzyme activity staining and Western blotting. Furthermore, the tyrosinase only synthesized in the cotyledons of dark-grown S. salsa seedlings, and declined with time in light, which was paralleled by the decreases of tyrosinase activity and betacyanin content in light. This result suggests that the specific tyrosinase is positively correlated with betalain biosynthesis, and betacyanin biosynthesis is induced by dark via synthesis of tyrosinase in S. salsa.
Seeds of the halophyte S. salsa were cultured in 24 h dark and 14 h blue light/10 h dark to examine the role of blue light and the blue-light absorbing photoreceptor cryptochrome 2 (CRY2) in betacyanin accumulation, hypocotyl elongation and cotyledon opening in S. salsa seedlings. Darkness significantly promoted betacyanin accumulation and hypocotyl elongation, but inhibited cotyledon opening. Blue light suppressed betacyanin accumulation and hypocotyl elongation, but stimulated cotyledon opening. Betacyanin in S. salsa seedlings decomposed with time in blue light. Western blot analysis showed that CRY2 protein accumulated both in hypocotyls and cotyledons of S. salsa seedlings grown in dark, but degraded with time in blue light, which was paralleled by a decrease of tyrosine hydroxylation activity of tyrosinase, a key enzyme involved in the betalain biosynthesis pathway. These results suggested CRY2 protein mediates betacyanin decomposition via inactivation of tyrosinase in S. salsa seedlings, and the blue-light-dependent degradation of CRY2 protein is crucial to its function.
研究结果表明:盐地碱篷红色素溶于水而不溶于纯有机溶剂、在酸性条件下稳定且为红色、在碱性条件下不稳定且为黄色、最大吸收峰为538 nm,根据这些特性我们初步确定该类红色素为甜菜红素(Betacyanins)。盐地碱篷甜菜红素在体外对高温、强光、碱和氧化剂敏感,而在低温、黑暗、酸性环境和没有氧化剂的条件下稳定。盐地碱篷甜菜红素体外清除超氧阴离子自由基的能力不如VC,而清除羟自由基的能力远高于VC。
光照抑制盐地碱蓬体内甜菜红素合成,黑暗、低温、高盐环境有利于盐地碱蓬甜菜红素积累,其中萌发期黑暗处理是诱导甜菜红素积累的最重要因子之一。盐地碱蓬种子暗培养3天得到的幼苗是紫红色,光照条件下培养3天得到的幼苗则是绿色。
用不同浓度的H_2O_2浇灌盐地碱蓬种子、喷洒5天苗龄盐地碱蓬幼苗的叶片、注射10天苗龄幼苗的叶片,测定其对盐地碱蓬甜菜红素含量的影响。结果显示不同方式的H_2O_2处理对盐地碱蓬不同器官的甜菜红素含量影响明显不同,H_2O_2浇灌后根中的H_2O_2含量增加最大,而叶片中甜菜红素含量增加最明显,但是叶片中的H_2O_2含量和根系中的甜菜红素含量增加却不显著。H_2O_2喷雾和注射虽然能明显提高叶片中的H_2O_2含量,但是对叶片甜菜红素含量却没有显著影响。器官中的H_2O_2含量和甜菜红素含量不成正比,叶片甜菜红素含量主要受到根系H_2O_2含量而不是叶片和茎H_2O_2含量的影响。因此,我们得出结论:H_2O_2浇灌引起的过氧化胁迫信号首先被根系感知,然后根系合成了某种可以传递到叶片并直接诱导甜菜红素合成的物质。
培养基中添加不同浓度的Ca~(2+)可以促进暗培养条件盐地碱蓬甜菜红素含量,钙螯合剂EGTA、离子通道抑制剂LaCl_3、CaM蛋白抑制剂CPZ和W7都可以抑制黑暗诱导的甜菜红素积累,但是这些处理对光培养条件下甜菜红素含量没有影响。黑暗和光照条件下盐地碱蓬甜菜红素含量都随内源钙的降低而降低。虽然CPZ和W7对光照条件CaM蛋白活性有抑制作用,但对光照条件下甜菜红素含量却没有明显影响。这些研究结果说明外源Ca~(2+)及CaM系统只参与了黑暗诱导的甜菜红素合成过程,内源钙同时参与了黑暗和光照条件下盐地碱蓬甜菜红素的合成过程。
在盐地碱蓬体内,酪氨酸酶活性只在黑暗条件下培养得到的明显积累甜菜红素的幼苗中检测到,而在光照条件下培养得到的幼苗中则检测不到,黑暗似乎是保持酪氨酸酶活性必需的条件。而且,当黑暗条件下培养得到的紫红色芽苗转入光照条件下后,酪氨酸酶的活性伴随着甜菜红素含量下降而逐渐降低。这说明酪氨酸酶参与了黑暗诱导的盐地碱蓬甜菜红素的合成。光照和黑暗调节盐地碱蓬甜菜红素合成和降解是通过调控酪氨酸酶活性实现的。
盐地碱蓬幼苗中CRY2蛋白在黑暗条件下积累,在蓝光条件下降解,这不仅和蓝光条件下甜菜红素含量下降的趋势一致,而且还和甜菜红素合成的关键酶酪氨酸酶的活性降低一致,这说明蓝光条件下CRY2蛋白在甜菜红素的降解过程中起着非常重要的作用,CRY2蛋白的降解是启动这种调控作用的关键。
As a halophyte, physiological and molecular responses of Suaeda salsa to salinity stress have been extensively studied. But nothing is known about the identification and the biosynthesis regulation of the red pigments in S. salsa. This paper focuses on the identification of the red pigments and the effects of abiotic stressors on their accumulation in S. salsa.
The results showed that these red pigments were insoluble in organic solvents but free in water, the pigments were red-violet and stable under acidic condition while yellow and unstable under alkaline condition, and they absorbed the highest value at wavelength near 538 nm. These results suggested that these red pigments in S. salsa are not anthocyanins but betacyanins.
The betacyanins extracted from S. salsa were sensitive to high temperature, high intensity of light, alkali and oxidant in vitro, and keep stable under low temperature, dark, acidic and free of oxidants conditions. The scavenging superoxide (O2-) ability of the betacyanins from S. salsa was lower than that of vitamin C, while the scavenging hydroxyl radical (OH·) ability of the betacyains was much higher than that of vitamin C in vitro.
Light suppressed betacyanin accumulation and enhanced their decomposition in vivo. Darkness, low temperature and high salinity enhanced betacyanin accumulation, and darkness at the germination phase is one of the most important environmental factors for the betacyanin accumulation in S. salsa.
Seeds, 5-d and 10-d old seedlings of C_3 halophyte S. salsa were watered, sprayed and infiltrated with 0, 0.10%, 0.33% and 1.00% H_2O_2 solution to examine whether H_2O_2 is involved in the betacyanin accumulation. H_2O_2 treatments led to the most significant betacyanin accumulation in the shoots of S. salsa seedlings when seeds were watered with H_2O_2 solution. The increase of betacyanin content was the highest and that of H_2O_2 content was the lowest in leaves, while the increase of betacyanin content was the lowest and that of H_2O_2 content was the highest in roots of seedlings obtained from seeds watered with H_2O_2 solution. When leaves of 5-d and 10-d old seedlings were sprayed and infiltrated with H_2O_2 solution, the H_2O_2 content was enhanced significantly, but the increase of betacyanin content was not notable in leaves, and the content of betacyanin and H_2O_2 was also at a lower level in roots. The H_2O_2 content in organs of S. salsa seedlings was not proportional to betacyanin content, and the betacyanin accumulation in leaves was correlated with the H_2O_2 production in roots rather than that in leaves and stems. These results suggested that the oxidative stress signal leading to betacyanin production in H_2O_2 treatments may be perceived by roots initially, then the signal was transferred to leaves; the signal transduction was performed and betacyanin accumulation was induced in leaves of S. salsa.
Seeds of S. salsa were cultured both in the dark and light for three days. Addition of Ca~(2+) in the 1/2 MS nutrient solution promoted betacyanin accumulation in the dark, and Ca~(2+ depletion by EGTA suppressed the darkness-induced betacyanin accumulation in shoots of S. salsa. Ca2+ channel blocker (LaCl_3) also inhibited the darkness-induced betacyanin accumulation. The highest activity of CaM and the maximum betacyanin content decreased by 51% and 45% in shoots of S. salsa seedlings treated with a potent CaM antagonist, chlorpromazine (CPZ), in the dark, respectively. Furthermore, another CaM antagonist, W7, also inhibited the activity of CaM and the dark-dependent betacyanin accumulation, whereas its less active structural analog, W5, had little effect on the responses to dark of S. salsa. These results suggest that Ca~(2+), Ca~(2+)-regulated ion channels and CaM play an important role in the darkness-induced betacyanin accumulation in shoots of C_3 halophyte S. salsa.
Seeds of the halophyte S. salsa were cultured in the dark for three days and betacyanin accumulation was induced significantly in cotyledons of S. salsa seedlings. Accumulated betacyanin in cotyledons of the dark-grown seedlings decomposed with time in light. As the betacyanin content declined, the hydroxylation and oxidation activity of tyrosinase extracted from cotyledons of the dark-grown seedlings decreased with time in light. The apparent molecular mass of tyrosinase in cotyledons of S. salsa seedlings was about 60 kDa estimated by SDS-PAGE, enzyme activity staining and Western blotting. Furthermore, the tyrosinase only synthesized in the cotyledons of dark-grown S. salsa seedlings, and declined with time in light, which was paralleled by the decreases of tyrosinase activity and betacyanin content in light. This result suggests that the specific tyrosinase is positively correlated with betalain biosynthesis, and betacyanin biosynthesis is induced by dark via synthesis of tyrosinase in S. salsa.
Seeds of the halophyte S. salsa were cultured in 24 h dark and 14 h blue light/10 h dark to examine the role of blue light and the blue-light absorbing photoreceptor cryptochrome 2 (CRY2) in betacyanin accumulation, hypocotyl elongation and cotyledon opening in S. salsa seedlings. Darkness significantly promoted betacyanin accumulation and hypocotyl elongation, but inhibited cotyledon opening. Blue light suppressed betacyanin accumulation and hypocotyl elongation, but stimulated cotyledon opening. Betacyanin in S. salsa seedlings decomposed with time in blue light. Western blot analysis showed that CRY2 protein accumulated both in hypocotyls and cotyledons of S. salsa seedlings grown in dark, but degraded with time in blue light, which was paralleled by a decrease of tyrosine hydroxylation activity of tyrosinase, a key enzyme involved in the betalain biosynthesis pathway. These results suggested CRY2 protein mediates betacyanin decomposition via inactivation of tyrosinase in S. salsa seedlings, and the blue-light-dependent degradation of CRY2 protein is crucial to its function.
引文
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