失水胁迫对月季切花花瓣内肽酶种类及其活性的影响
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摘要
本试验以探讨“失水胁迫对花瓣内肽酶活性和种类的诱导及其对月季切花失水胁迫耐性的影响”为目的。首先选用耐失水品种‘Samantha’和中度耐失水品种‘Belami’,并且以创造不同的胁迫程度为目的,分别进行了带枝花和离枝花两种方式下的失水胁迫处理,采用分光光度法测定了内肽酶的活性与种类,在此基础上,以‘Samantha’为试材,进行反复失水胁迫处理,通过底物胶电泳法进一步探讨了失水胁迫对内肽酶活性和种类的诱导。
1、两品种之间的比较研究表明:
1) 在0～60h失水胁迫过程中,带枝花花枝复水率出现由缓慢下降到快速下降的拐点,‘Samantha’(30h)晚于‘Belami’(24h);两品种花朵水势对应于复水率拐点呈现高度吻合的由快速下降转向缓慢下降的转折点。
2) 两品种花瓣内肽酶活性诱导上升的转折点与其相应的复水率迅速降低的拐点相一致。胁迫诱导引起的花瓣中金属蛋白酶和丝氨酸蛋白酶活性的变化均是‘Samantha’显著低于‘Belami’。
离枝花获得了与带枝花的一致的结果。
由此说明,切花月季品种间失水胁迫耐性差异可能与胁迫对花瓣中金属蛋白酶和丝氨酸蛋白酶活性诱导的差异有关。对‘Samantha’的诱导显著低于‘Belami’,这可能是‘Samantha’较‘Belami'耐失水的原因之一。
2、‘Samantha’反复失水胁迫处理结果表明:
1) 失水胁迫24h后,经6h复水,花颈弯曲状况能够恢复到正常状态,同时花朵水势能够恢复到胁迫前的水平,但是花枝鲜重和花瓣内肽酶活性却在复水12h才基本上恢复到胁迫前水平;再次失水胁迫,花枝形态和花朵水分状况都表现出与第一次胁迫相近的变化,但是花瓣内肽酶活性较第一次胁迫过程中的变化更剧烈,上升幅度更大。
2) 花瓣中丝氨酸蛋白酶活性的迅速增加与切花的衰老紧密联系;失水胁迫强烈诱导了丝氨酸蛋白酶活性,失水胁迫18h,其活性就和自然衰老过程中萎蔫期(6级)时的相同,复水6h仍不能明显下降,再次失水胁迫6h其活性就已经达到萎蔫期(6级)时的水平;ABA预处理减轻了失水胁迫的征状,水势的降低、鲜重的损失及内肽酶活性的水平都显著被抑制。
3) 试验中对月季切花花瓣内肽酶底物胶电泳测定方法进行了优化,结果表明,花瓣蛋白粗提液通过Sephadex G-25柱子去盐去色素等杂质后,在40℃下前处理10min,然后用12%分离胶(含有0.15%明胶)进行分离,复性完成后温育16h,内肽酶的酶解条带在蓝黑色的凝胶背景上清晰出现。
4) 底物胶电泳法结果显示,失水胁迫诱导的内肽酶与衰老相关的内肽酶谱带相同,共有3条,分子量从大到小依次大约为200 kDa、123.5 kDa、97.4 kDa,分别命名为EP1、EP2、EP3,其中EP2、EP3与衰老关系更为密切。EP1、EP2、EP3的最适反应温度是42℃;EP1的适宜pH在酸性到中性范围内,EP2和EP3在中性到碱性范围内,pH7时三者活性都能明显表现;EP2和EP3是丝氨酸蛋白酶,EP1可能是金属蛋白酶。
This work was aimed to investigate induction of the activity and types of endoproteases (EP) related to tolerance to water deficit stress (WDS) in cut rose (Rosa hybrida) during WDS. Two cultivars, 'Samantha' with the strong tolerance to WDS, and 'Belami', with the medium one, were used as materials. To make different process of WDS, the two cultivars were treated separately with two manners, namely flowers with or without branches, and the activity and types of EP were determined based on the spectrophotometer method. 'Samantha' was furthermore used to probe into the induction of the activity and types of EP by repeated WDS treatment through gelatin-SDS-PAGE method.1. Based on comparison of the two cultivars under WDS:1) A turning point, in which recovery rate of flowers was going to be decreasing rapidly, was observed in flowers with branches, and the occurrence of the turning point was later in 'Samantha'(30 h) than in 'Belami'(24 h). The turning point of quick falling of water potential (WP) of flower to slow falling was also observed in the flowers of both cultivar, these points were high identical with the turning points of their recovery rate.2) WDS-induced increase of EP activity was later in 'Samantha' than in 'Belami'. And the turning points of quick increase of EP activity induced by WDS in both cultivars were consistent with the turning points of their recovery rates. Changes of both Metallo-proteinase and serine proteinase activities of petals induced by WDS were significantly smaller in 'Samantha' than in 'Belami'.The experiments with the flowers without branches showed substantially the same results as those described above with the flowers with branches.These results above suggested that the difference of tolerance to WDS between different cultivars might be related to the different changes of metallo-proteinase and serine proteinase activities in petals induced by WDS.2. Based on the results of'Samantha' under repeated WDS treatment:1) WR for 6 h after WDS for 24 h, the bend states of flower neck could recover to the normal, at the same time, the WP of flower could come back to the level of no stress. But, the fresh weight of branch and the EP activity of petals could not get back to a basal low level as that of before WDS treatment until WR for 12 h. When WDS being given again, the similar changes took on in flower appearance, flower and branch water conditions, while more acutely change presented in EP activity of petals.2) The quick increase of serine proteinase activity in petals was correlative with senescence. The activity was induced strongly by WDS. WDS for 18 h, the activity was the same as that of senescence (stage 6) and did not decreased significantly until WR for 6 h. WDS for 6 h was given again, the activity came to the level of senescence. The WDS-inducible appearance of flower was weaken by ABA; the falling of water potential, the loss of fresh weight and the hoist of EP activity were controlled.3) The determining method of gelatin-containing polyacrylamide gels in EP from petals of cut rose was optimized. The result showed that removing salts and pigments from crude extracts through column of Sephadex G-25 was vital to the preparation of protein sample. The prepared samples were treated for 10
min at 40 ℃, loaded in 12 % separating gel containing 0.15 % gelatin, following electrophoresis, incubated for 16 h, Clear bands were observed on the dark blue background.4) The results of gelatin-SDS-PAGE showed that the number of bands induced by WDS was the same as that of senescence-related EPs. According to the molecular weight from large to small, named EP1(200 kDa), EP2(123.5 kDa), EP3(97.4 kDa) respectively. Among them, EP2 and EP3 were most correlative with senescence. The optimum activation temperature of EPl, EP2 and EP3 was 42 ℃; As for the optimum activation pH, EPl had obvious activity from acidic to neutral, EP2 and EP3 had clear activity from neutral to alkali, and all the three had obvious activity was pH 7; EP2 and EP3 were serine proteinases, while EP1 was metallo-proteinase.
1、两品种之间的比较研究表明:
1) 在0～60h失水胁迫过程中,带枝花花枝复水率出现由缓慢下降到快速下降的拐点,‘Samantha’(30h)晚于‘Belami’(24h);两品种花朵水势对应于复水率拐点呈现高度吻合的由快速下降转向缓慢下降的转折点。
2) 两品种花瓣内肽酶活性诱导上升的转折点与其相应的复水率迅速降低的拐点相一致。胁迫诱导引起的花瓣中金属蛋白酶和丝氨酸蛋白酶活性的变化均是‘Samantha’显著低于‘Belami’。
离枝花获得了与带枝花的一致的结果。
由此说明,切花月季品种间失水胁迫耐性差异可能与胁迫对花瓣中金属蛋白酶和丝氨酸蛋白酶活性诱导的差异有关。对‘Samantha’的诱导显著低于‘Belami’,这可能是‘Samantha’较‘Belami'耐失水的原因之一。
2、‘Samantha’反复失水胁迫处理结果表明:
1) 失水胁迫24h后,经6h复水,花颈弯曲状况能够恢复到正常状态,同时花朵水势能够恢复到胁迫前的水平,但是花枝鲜重和花瓣内肽酶活性却在复水12h才基本上恢复到胁迫前水平;再次失水胁迫,花枝形态和花朵水分状况都表现出与第一次胁迫相近的变化,但是花瓣内肽酶活性较第一次胁迫过程中的变化更剧烈,上升幅度更大。
2) 花瓣中丝氨酸蛋白酶活性的迅速增加与切花的衰老紧密联系;失水胁迫强烈诱导了丝氨酸蛋白酶活性,失水胁迫18h,其活性就和自然衰老过程中萎蔫期(6级)时的相同,复水6h仍不能明显下降,再次失水胁迫6h其活性就已经达到萎蔫期(6级)时的水平;ABA预处理减轻了失水胁迫的征状,水势的降低、鲜重的损失及内肽酶活性的水平都显著被抑制。
3) 试验中对月季切花花瓣内肽酶底物胶电泳测定方法进行了优化,结果表明,花瓣蛋白粗提液通过Sephadex G-25柱子去盐去色素等杂质后,在40℃下前处理10min,然后用12%分离胶(含有0.15%明胶)进行分离,复性完成后温育16h,内肽酶的酶解条带在蓝黑色的凝胶背景上清晰出现。
4) 底物胶电泳法结果显示,失水胁迫诱导的内肽酶与衰老相关的内肽酶谱带相同,共有3条,分子量从大到小依次大约为200 kDa、123.5 kDa、97.4 kDa,分别命名为EP1、EP2、EP3,其中EP2、EP3与衰老关系更为密切。EP1、EP2、EP3的最适反应温度是42℃;EP1的适宜pH在酸性到中性范围内,EP2和EP3在中性到碱性范围内,pH7时三者活性都能明显表现;EP2和EP3是丝氨酸蛋白酶,EP1可能是金属蛋白酶。
This work was aimed to investigate induction of the activity and types of endoproteases (EP) related to tolerance to water deficit stress (WDS) in cut rose (Rosa hybrida) during WDS. Two cultivars, 'Samantha' with the strong tolerance to WDS, and 'Belami', with the medium one, were used as materials. To make different process of WDS, the two cultivars were treated separately with two manners, namely flowers with or without branches, and the activity and types of EP were determined based on the spectrophotometer method. 'Samantha' was furthermore used to probe into the induction of the activity and types of EP by repeated WDS treatment through gelatin-SDS-PAGE method.1. Based on comparison of the two cultivars under WDS:1) A turning point, in which recovery rate of flowers was going to be decreasing rapidly, was observed in flowers with branches, and the occurrence of the turning point was later in 'Samantha'(30 h) than in 'Belami'(24 h). The turning point of quick falling of water potential (WP) of flower to slow falling was also observed in the flowers of both cultivar, these points were high identical with the turning points of their recovery rate.2) WDS-induced increase of EP activity was later in 'Samantha' than in 'Belami'. And the turning points of quick increase of EP activity induced by WDS in both cultivars were consistent with the turning points of their recovery rates. Changes of both Metallo-proteinase and serine proteinase activities of petals induced by WDS were significantly smaller in 'Samantha' than in 'Belami'.The experiments with the flowers without branches showed substantially the same results as those described above with the flowers with branches.These results above suggested that the difference of tolerance to WDS between different cultivars might be related to the different changes of metallo-proteinase and serine proteinase activities in petals induced by WDS.2. Based on the results of'Samantha' under repeated WDS treatment:1) WR for 6 h after WDS for 24 h, the bend states of flower neck could recover to the normal, at the same time, the WP of flower could come back to the level of no stress. But, the fresh weight of branch and the EP activity of petals could not get back to a basal low level as that of before WDS treatment until WR for 12 h. When WDS being given again, the similar changes took on in flower appearance, flower and branch water conditions, while more acutely change presented in EP activity of petals.2) The quick increase of serine proteinase activity in petals was correlative with senescence. The activity was induced strongly by WDS. WDS for 18 h, the activity was the same as that of senescence (stage 6) and did not decreased significantly until WR for 6 h. WDS for 6 h was given again, the activity came to the level of senescence. The WDS-inducible appearance of flower was weaken by ABA; the falling of water potential, the loss of fresh weight and the hoist of EP activity were controlled.3) The determining method of gelatin-containing polyacrylamide gels in EP from petals of cut rose was optimized. The result showed that removing salts and pigments from crude extracts through column of Sephadex G-25 was vital to the preparation of protein sample. The prepared samples were treated for 10
min at 40 ℃, loaded in 12 % separating gel containing 0.15 % gelatin, following electrophoresis, incubated for 16 h, Clear bands were observed on the dark blue background.4) The results of gelatin-SDS-PAGE showed that the number of bands induced by WDS was the same as that of senescence-related EPs. According to the molecular weight from large to small, named EP1(200 kDa), EP2(123.5 kDa), EP3(97.4 kDa) respectively. Among them, EP2 and EP3 were most correlative with senescence. The optimum activation temperature of EPl, EP2 and EP3 was 42 ℃; As for the optimum activation pH, EPl had obvious activity from acidic to neutral, EP2 and EP3 had clear activity from neutral to alkali, and all the three had obvious activity was pH 7; EP2 and EP3 were serine proteinases, while EP1 was metallo-proteinase.
引文
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