外源γ-氨基丁酸对盐碱胁迫下甜瓜种子萌发的影响 | [PDF全文] |
Salt-sensitive variety of melon (Cucumis melo L.) seeds (cv “Yipintianxia 208”), which is sensitive to salinity-alkalinity stresses, was chosen as experiment material. The effects of different concentrations (0, 5, 10, and 50 mmol/L) of exogenous GABA on germination index, radicle antioxidant enzyme activities, and GABA metabolism of melon seeds under 50 mmol/L salinity-alkalinity stress conditions (NaCl∶Na2SO4∶ NaHCO3∶Na2CO3 as molar volume ratio 1∶9∶9∶1) were studied. There were five treatments in this experiment (i) CK: purified water presoaking and pre-germination; (ii) T1: purified water presoaking and pre-germination under 50 mmol/L salinity-alkalinity stress; (iii) T2: 5 mmol/L GABA presoaking and pre-germination under 50 mmol/L salinity-alkalinity stress; (iv) T3: 10 mmol/L GABA presoaking and pre-germination under 50 mmol/L salinity-alkalinity stress; (v) T4: 50 mmol/L GABA presoaking and pre-germination under 50 mmol/L salinity-alkalinity stress.
The results showed that salinity-alkalinity stresses significantly inhibited seeds germination and the growth of radicles and embryos of melon. Soaking seeds in different concentrations of GABA partly decreased the stress-induced inhibition on seeds germination of melon. Compared with the melon seeds in the treatment of salinity-alkalinity stress without GABA, a presoaking concentration of 5 mmol/L GABA improved superoxide dismutase (SOD) activity, and presoaking with 10 mmol/L GABA improved soluble protein content, endogenous GABA content and glutamate decarboxylase (GAD) activity in the radicles; presoaking with 50 mmol/L GABA improved seed germination rates, germination potential, vigor index, fresh mass, lengths of radicles and embryos, and peroxidase (POD), catalase (CAT) and GABA transaminase (GABA-T) activities and decreased malondialdehyde (MDA) content.
It was concluded that 50 mmol/L GABA presoaking significantly alleviate the inhibition of melon seeds under salinity-alkalinity stress, and improved the salt tolerance of melon seeds.
甜瓜 ( Cucumis melo L.)是设施栽培的重要果蔬作物,因发育周期短、供给时间长、高产优质等特点,是我国农业栽培的重要作物之一,也是西北地区主要栽培作物之一,中国甜瓜产量约占世界总产量的50%[1]。我国的盐渍土总面积约3.6×107 hm2,主要分布在西北、华北及东北等地区,严重影响着作物的产量、品质和效益。土壤盐碱胁迫严重抑制、延迟甚至阻碍了大多数植物种子的萌发[2]。因此,如何利用好大面积的次生盐渍化土壤继续维持和发展果蔬生产,已经成为研究人员亟待解决的问题。
γ-氨基丁酸(γ-aminobutyric acid,GABA)作为自由态的四碳非蛋白质氨基酸,普遍存在于动植物组织中,是一种主要的胞内信号分子[3]。在动物体内,GABA是一种重要的抑制性神经传导物质,是最普通的脑内抑制性递质[4]。在高等植物体内,当植物遭到生物与非生物胁迫时,通常会在生理和生化等不同水平作出响应,产生多种逆境耐受机制,以便在胁迫条件下得以生存[5]。研究表明,外源GABA可以通过提高抗氧化酶活性,降低活性氧水平及膜脂过氧化程度,维持较高的光合系统Ⅱ活性,促进幼苗的生长及生物量积累,缓解NaCl胁迫对番茄种子的伤害[6];外源GABA能通过调节活性氧代谢,缓解低氧胁迫对甜瓜种子萌发造成的伤害[7]。然而,关于不同摩尔浓度GABA对盐碱胁迫下甜瓜种子萌发影响的研究尚鲜有报道。因此,本试验以对盐碱敏感的甜瓜(Cucumis melo L)品种“一品天下208”为试材[8],研究不同摩尔浓度GABA溶液浸种对盐碱胁迫下甜瓜种子萌发能力和内部生理指标的影响,旨在筛选出能缓解盐碱胁迫对甜瓜种子萌发抑制作用的最适GABA浓度。
1 材料与方法 1.1 试验材料以盐碱敏感品种“一品天下208”[8]种子为试验材料。
1.2 试验方法试验于西北农林科技大学园艺学院试验室内进行,共设5个处理:1)蒸馏水浸种催芽(对照,CK);2)蒸馏水浸种+50 mmol/L盐碱胁迫[c(NaCl)∶c(Na2SO4)∶c(NaHCO3)∶c(Na2CO3)=1∶ 9∶ 9∶ 1](处理1,T1);3)5 mmol/L GABA 浸种+50 mmol/L盐碱胁迫(处理2,T2);4)10 mmol/L GABA 浸种+50 mmol/L盐碱胁迫(处理3,T3);5)50 mmol/L GABA 浸种+50 mmol/L盐碱胁迫(处理4,T4)。选取大小一致,颗粒饱满的甜瓜种子经温汤浸种后,置于含有不同摩尔浓度GABA溶液的烧杯中,并于28 ℃的人工气候箱内黑暗浸种6 h,然后将种子置于直径9 cm铺有3层滤纸的培养皿中,分别滴加相应摩尔浓度GABA溶液(对照和盐碱胁迫处理滴加蒸馏水)浸湿滤纸,每个培养皿内放入20粒种子,在黑暗条件下28 ℃人工气候箱催芽,每个处理3次重复。培养期间每天补充适量的去离子水或相应摩尔浓度GABA水溶液,以保证水分与GABA浓度恒定。以开始露出胚根作为发芽标准。每天记录发芽种子数,在催芽第5天收集胚根和胚芽,进行相关指标的测定。
1.3 测定项目与方法 1.3.1 萌发指标的测定
${\rm{发芽率}} = \frac{第5天发芽种子总数}{总供试种子数} \times 100\% . $
${\rm{发芽势}} = \frac{第3天发芽种子总数}{总供试种子数} \times 100\% . $
活力指数(vigor index,VI)=S×∑(Gt/Dt),式中:S为新苗总鲜质量,g;Gt为总供试种子在t时间之内萌发的数量;Dt为种子萌发所对应的时间.
胚芽和胚根长度的测定:利用扫描仪(Epson Experssion 1680)获取芽苗影像,然后使用Image J软件分析胚芽和胚根长度。
1.3.2 生理指标的测定超氧化物歧化酶(superoxide dismutase,SOD)活性测定参考GIANNOPOLITIS等[9]的方法,以抑制氮蓝四唑(nitrobluetetrazolium,NBT)光化还原的50%作为一个酶活性单位;过氧化物酶(peroxidase,POD)活性测定参考曾韶西等[10]方法;过氧化氢酶(catalase,CAT)活性测定参考DANIELS等[11]方法;丙二醛含量测定参照HEATH等[12]的方法;可溶性蛋白质含量测定选用考马斯亮蓝G-250染色法参照BRADFORD等[13]方法;GABA含量测定参照ZHANG等[14]的方法;谷氨酸脱羧酶(glutamate decarboxylase,GAD)活性参照沈黎明等[15]和JOHNSON等[16]的方法测定;GABA转氨酶(GABA transaminase,GABA-T)活性参照ANSARI等[17]的方法。
1.4 数据分析用统计分析软件SAS 8.1进行试验数据处理和分析。采用单因素方差(One-way ANOVA)分析及Duncan多重比较(Duncan’s multiple range test)进行差异显著性分析(α=0.05)。
2 结果与分析 2.1 对发芽率、发芽势和活力指数的影响由图1可以看出,盐碱胁迫处理显著降低了甜瓜种子的发芽率和发芽势,外源GABA浸种处理对甜瓜种子的发芽率和发芽势无显著影响,但50 mmol/L GABA浸种处理的种子发芽率和发芽势均高于单纯盐碱胁迫处理。与对照相比,单纯盐碱胁迫显著降低了甜瓜种子的活力指数;而5 mmol/L和50 mmol/L GABA浸种处理可以增加盐碱胁迫下甜瓜种子的活力指数,分别比单纯盐碱胁迫增加了26.18%和26.60%.
由表1可以看出,与对照相比,单纯盐碱胁迫处理显著降低了甜瓜芽苗总鲜质量和抑制了胚根和胚芽生长。不同摩尔浓度GABA浸种处理减缓了盐碱胁迫对上述指标的抑制作用,其中50 mmol/L GABA浸种处理的总鲜质量、胚根和胚芽生长效果最好,分别比单纯盐碱胁迫处理增加了40.87%、54.63%和28.72%。
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由图2可以看出,单纯盐碱胁迫处理显著增加了甜瓜胚根内MDA质量摩尔浓度,比对照增加了36.44%。在盐碱胁迫下,甜瓜胚根内MDA含量表现出随GABA浸种浓度的增加呈逐渐降低的变化趋势,当摩尔浓度为50 mmol/L GABA时,甜瓜胚根内MDA含量最低。
由图3可以看出,单纯盐碱胁迫处理增加了甜瓜芽苗胚根内可溶性蛋白质含量;外源GABA浸种处理后胚根内可溶性蛋白质含量随着GABA摩尔浓度的增加表现出先升高后降低的变化趋势,在GABA摩尔浓度为10 mmol/L时胚根内可溶性蛋白质含量最高。
由图4可知,盐碱胁迫处理显著增加了甜瓜芽苗胚根内SOD和POD活性,降低了CAT活性;外源GABA浸种处理的甜瓜胚根内SOD活性表现出先升高后降低的变化趋势,在GABA摩尔浓度为5 mmol/L时活性最大;CAT活性随着GABA摩尔浓度的升高呈现出逐渐升高的变化趋势,在GABA摩尔浓度为50 mmol/L时活性最大。盐碱胁迫下,胚根内POD活性表现出随GABA摩尔浓度的增加逐渐升高的变化趋势,在GABA摩尔浓度为50 mmol/L时胚根内POD活性达到最大。
由图5可以看出,在盐碱胁迫条件下甜瓜芽苗胚根内GABA含量与对照相比显著增加了15.07%。不同外源GABA浸种处理后,胚根内GABA含量表现出随外源GABA摩尔浓度的增加而先升高后降低的变化趋势,10 mmol/L GABA处理的胚根内GABA含量比单纯盐碱胁迫增加了41.06%。
由图6可以看出,盐碱胁迫处理显著降低了甜瓜芽苗胚根内GAD活性,仅为对照的27.52%。在盐碱胁迫下,不同摩尔浓度的外源GABA浸种处理后,胚根内GAD活性表现出随GABA摩尔浓度的增加先升高后降低的变化趋势,10 mmol/L GABA浸种处理的胚根内GAD活性最高。盐碱胁迫处理显著降低了甜瓜芽苗胚根内GABA-T活性,比对照降低了39.68%;在盐碱胁迫条件下采用不同浓度的外源GABA浸种后,胚根内GABA-T活性随着GABA摩尔浓度的升高表现出逐渐升高的变化趋势,在GABA摩尔浓度为50 mmol/L时胚根内GABA-T活性最高。
盐碱胁迫,降低了植物光合作用,抑制了植物正常生长发育[8, 18]。罗黄颖等[6]发现外源GABA能显著缓解高盐胁迫下番茄种子的萌发。本试验结果中盐碱胁迫显著降低了甜瓜种子的发芽率、发芽势、活力指数、总鲜质量,并且抑制了胚根和胚芽生长,外源GABA浸种缓解了盐碱胁迫对甜瓜芽苗总鲜质量、胚根和胚芽生长的抑制作用,50 mmol/L外源GABA浸种的效果最佳。逆境胁迫下植物易发生膜脂过氧化使体内自由基代谢失衡,MDA含量升高损坏细胞结构和功能[19]。本试验在单纯盐碱胁迫下胚根内MDA含量显著增加,50 mmol/L的GABA浸种能明显降低胚根内MDA含量。
抗氧化酶是植物体内活性氧清除系统,主要维持活性氧代谢平衡,保证膜结构功能完整。研究显示,植物受非生物胁迫时,SOD迅速催化细胞中多余的O2-转化成H2O2和O2[20, 21],POD和CAT则负责清除过多的H2O2。周翔等[22]研究表明,外源GABA能提高逆境胁迫下玉米体内的抗氧化酶活性[23]。逆境胁迫也能诱导植物体内GABA迅速积累[24],提高植物体抗氧化酶活性缓解膜脂过氧化对植物体的伤害。本试验结果,盐碱胁迫显著增加了胚根内SOD和POD活性,降低了CAT活性,这与贺岩等[25]和高永生等[26]研究结果不同,这可能与植物种类和测量部位不同相关。在盐碱胁迫下外源GABA浸种后,胚根内SOD、POD、CAT活性均发生显著变化,在GABA摩尔浓度分别为5 mmol/L、50 mmol/L和50 mmol/L时胚根内SOD、CAT和POD活性最高,这与罗黄颖等[6]研究的适当施加外源GABA能增加植物体内抗氧化酶的活性一致。
在高等植物体内,GABA在传递信号,调节pH值,应答胁迫反应和参与碳氮代谢等方面,扮演重要的角色[27]。本试验中,盐碱胁迫下胚根内GABA含量显著升高,与逆境胁迫可以诱发植物体内GABA的快速积累一致[28]。而胚根内GAD活性在单纯盐碱胁迫下显著降低,与盐碱胁迫下GAD活性被激活以提高植物的抗性[29]不同,这可能与植物所处的pH环境以及Ca2+/CaM有关。10 mmol/L GABA浸种时GABA含量和GAD活性显著升高,50 mmol/L GABA浸种时GABA-T活性最高,加快了GABA向琥珀酸半醛降解参与三羧酸循环的速度,提高甜瓜种子的耐盐碱能力。
以上所述,盐碱胁迫显著抑制了甜瓜种子的萌发、胚根和胚芽的生长;不同摩尔浓度GABA浸种部分缓解了盐碱胁迫对甜瓜种子的抑制作用。50 mmol/L的GABA浸种能提高种子的发芽率、发芽势、活力指数、总鲜质量,并增加胚根和胚芽长度,还能提高POD、CAT和GABA-T活性,降低MDA含量,显著缓解了盐碱胁迫对甜瓜种子萌发期的伤害。
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