植物生长调节剂是增强植株抗旱能力的辅助方法之一[6]。褪黑素（melatonin,MT）化学名为N-乙酰-5-甲氧基色胺,是一类广泛存在于动植物体内的重要吲哚类激素[7]。有研究[7]发现,褪黑素可缓解干旱胁迫对植物膜系统的损伤,还可促进干旱胁迫下植物叶片中还原型抗坏血酸（AsA）和还原型谷胱甘肽（GSH）含量增加。研究[8]表明,褪黑素可在干旱胁迫下帮助植物稳定AsA-GSH循环,这是植物体内重要的抗氧化防御机制,同时增强AsA-GSH循环清除ROS的能力[9],AsA-GSH循环中重要的抗氧化物质AsA和GSH是植物体中为数不多的既可以直接还原ROS,又可以作为酶底物清除ROS的抗氧化物质[10],在经过褪黑素处理后二者含量均有显著提升。
近年来有关褪黑素的相关研究逐渐增多,但关于褪黑素对干旱胁迫下大豆AsA-GSH循环影响的研究还鲜有报道。因此本试验探究褪黑素处理下大豆AsA-GSH循环的变化与特征,为利用褪黑素抵抗逆境的实际应用提供理论依据。
1 材料与方法
1.1 试验设计
以干旱敏感型大豆品种绥农26为供试品种,盆栽桶直径30.0cm、高33.0cm。为了有效控制土壤含水量和避免土壤板结,采用蛭石、珍珠岩和黑钙土体积比1:3:12混合作为基质。混合培养土理化性质：有机质3.9mg/kg、碱解氮72.1mg/kg、速效磷14.1mg/kg、速效钾173.0mg/kg、pH 7.1。施肥量按照大田磷酸二铵300kg/hm2、尿素75kg/hm2和硫酸钾75kg/hm2进行折算施用。播种前测量每盆基质质量和含水量,计算出基质干重。通过环刀法测量最大持水量,以便后续控水过程中准确掌握田间持水量。
试验于2020年在黑龙江省大庆市国家杂粮工程技术研究中心进行。为了避免雨水淋浇,所有盆栽均放置在遮雨棚中。大豆种子采用75%的酒精处理2min,再用5% NaClO消毒5min,用无菌水洗净后播种。每盆播种6粒,均匀排布。在子叶期（VC）定苗3株,去除过大或过小的幼苗,保留长势均匀适中的幼苗。
自播种至鼓粒始期,采用称重法控制土壤含水量为田间持水量的80%。试验设正常供水（CK）、干旱（D）和干旱胁迫喷施褪黑素（D+M）3个处理,每个处理100盆。正常供水处理（CK）：保持土壤含水量为田间持水量的80%;干旱胁迫处理（D）：停止供水并于每天18:00称重,土壤含水量达到田间持水量的50%为止,之后补水维持此含水量,第3次取样结束后恢复供水至田间持水量的80%,直至收获;干旱胁迫喷施褪黑素处理（D+M）：土壤含水量达到田间持水量的50%后,连续3d在21:00喷施浓度100μmol/L的褪黑素。
1.2 测定项目与方法
1.2.1 植株形态指标及其干、鲜重 将植株取出后洗净,将地上部和地下部分开,测量株高、根长、茎粗和根体积等形态指标。
1.2.3 AsA-GSH循环指标 参照Cakmak等[15]的方法采用过氧化氢法测定抗坏血酸过氧化物酶（APX）活性。
1.2.4 取样时间及方法 用于生理指标和干物质测定的样品分别在鼓粒初期褪黑素处理后的第5、10、15天取样;用于酶活性等其他生理指标测定的样品用液氮冷冻并快速转移至-80℃冰箱保存待测。
1.3 数据处理
用SPSS软件的Duncan’s多重比对法进行统计分析,用Origin软件绘图。
2 结果与分析
2.1 褪黑素对干旱胁迫下大豆ROS副产物的影响
由图1可知,O2-. 和H2O2含量随着干旱胁迫时间的延长逐渐提高,与CK处理相比,在处理后15d,D处理O2-. 和H2O2含量显著提高,且在15d达到最大值,分别提高了98.43%和95.05%;与D处理相比,D+M处理O2-. 含量在5、10和15d时分别下降了7.67%、15.37%和18.09%,H2O2含量分别下降了18.30%、21.71%和17.73%,在15d时3个处理间达到差异显著水平。
图1
图1
褪黑素对干旱胁迫下大豆ROS副产物的影响
不同小写字母代表差异显著（P < 0.05）,下同
Fig.1
Effects of melatonin on ROS by-products of soybean under drought stress
Different lowercase letters represent significant difference (P < 0.05), the same below
2.2 褪黑素对干旱胁迫下大豆AsA-GSH循环关键酶活性的影响
由图2可知,与CK处理相比,D处理APX活性显著上升,处理后5、10和15d,APX活性分别提高了33.11%、43.08%和41.59%;在处理后10d,D处理相比CK处理GR活性提高了64.63%,达到差异显著水平。与D处理相比,D+M处理APX和GR活性显著提高,D+M处理相比D处理的APX活性在第5、10和15d时分别提高了27.27%、28.13%和32.37%,GR活性分别提高了70.10%、14.50%和9.00%。
图2
图2
褪黑素对干旱胁迫下大豆AsA-GSH循环关键酶活性的影响
Fig.2
Effects of melatonin on key enzymes activities of AsA-GSH cycle in soybean under drought stress
2.3 褪黑素对干旱胁迫下大豆AsA-GSH循环物质含量的影响
AsA、GSH、GSSG和DHA是植物AsA-GSH循环的主要成分,并且有调节AsA-GSH循环的重要功能。由图3可知,与CK处理相比,D处理的AsA、GSH和GSSG含量显著上升,处理后10d时上升幅度最大,分别为30.71%、32.70%和32.70%,DHA含量在处理后15d时达到差异显著水平。与D处理相比,D+M处理进一步促进了AsA、GSH和GSSG含量的提升,处理后10d分别提高了22.39%、15.30%和15.30%,达到差异显著水平。DHA含量在处理后15d时达到差异显著水平
图3
图3
褪黑素对干旱胁迫下大豆AsA-GSH循环物质含量的影响
Fig.3
Effects of melatonin on AsA-GSH cycle substances contents in soybean under drought stress
2.4 褪黑素对干旱胁迫下大豆形态指标的影响
由表1可知,大豆鼓粒期遭遇干旱胁迫造成株高显著降低,茎粗和底荚高有所下降。与CK处理相比,D处理的株高、茎粗和底荚高分别减少了4.61%、5.88%和10.26%。与D处理相比,D+M处理的株高、茎粗和底荚高分别提高了1.80%、2.06%和2.31%。
表1 褪黑素对干旱胁迫下大豆形态指标的影响
Table 1
| 处理 Treatment | 株高 Plant height (cm) | 茎粗 Stem diameter (mm) | 底荚高 Bottom pod height (cm) |
|---|---|---|---|
| CK | 91.45±0.45a | 7.20±0.25a | 21.50±1.03a |
| D | 87.42±3.12b | 6.80±0.21a | 19.50±0.70a |
| D+M | 88.99±2.36ab | 6.94±0.19a | 19.95±1.12a |
不同小写字母代表差异显著（P < 0.05）,下同
Different lowercase letters indicate significant difference (P < 0.05), the same below
2.5 褪黑素对干旱胁迫下大豆产量构成因素的影响
由表2可知,与CK处理相比,D处理的单株荚数、单株粒数、百粒重和单株粒重显著下降,下降幅度分别为10.97%、7.11%、29.64%和33.75%。与D处理相比,D+M处理上述指标均显著增加,分别增加了4.68%、4.18%、12.20%和9.20%。
表2 褪黑素对干旱胁迫下大豆产量构成因素的影响
Table 2
| 处理 Treatment | 单株荚数 Pod number per plant | 单株粒数 Grain number per plant | 百粒重 100-seed weight (g) | 单株粒重 Grain weight per plant (g) |
|---|---|---|---|---|
| CK | 17.75±0.14a | 38.84±0.25a | 19.13±0.71a | 9.00±0.40a |
| D | 15.99±0.26c | 36.26±0.33c | 14.75±0.48c | 6.23±0.18c |
| D+M | 16.74±0.14b | 37.78±0.19b | 16.55±0.49b | 6.80±0.52b |
3 讨论
干旱胁迫会导致大豆鼓粒期的生长发育受阻,从而造成形态指标以及干物质累积量的降低[19]。本研究中,干旱胁迫导致大豆株高、茎粗和底荚高下降,而经过褪黑素处理后,株高、茎粗和底荚高显著上升,说明褪黑素能有效缓解干旱胁迫对大豆植株形态发育的抑制。鼓粒期是大豆生殖生长的关键时期,此时期遭遇干旱胁迫会导致大豆产量降低[20]。研究[21]表明,褪黑素可通过提高抗氧化酶活性和抗氧化剂含量,进而减少植物体内ROS含量,减轻植物膜脂过氧化程度。H2O2和O2-. 是植物体内ROS的副产物,可在一定程度上体现植物体内ROS的富集程度,而干旱胁迫会抑制大豆的正常生长发育,破坏植物体内ROS平衡的稳定状态,造成活性氧物质大量外泄,过多的活性氧自由基不能被及时清除,会加快膜脂过氧化进程,降低膜系统的完整性,损伤蛋白质,最终导致植物体内生理生化代谢紊乱,抑制植物生长发育[22]。本研究发现,鼓粒期干旱胁迫导致大豆叶片中O2-. 和H2O2含量显著增加,与正常供水处理相比,干旱胁迫处理O2-. 和H2O2含量显著提高。研究[21]发现,褪黑素可提高大豆在逆境胁迫下的耐受性,原因可能是褪黑素可帮助大豆降低植物体内的活性氧自由基。本试验研究表明,褪黑素可有效减少干旱胁迫下大豆叶片内O2-. 和H2O2的积累,说明褪黑素对减少ROS副产物和稳定ROS平衡具有重要作用。
AsA-GSH循环是植物体内重要的抗氧化系统之一[23,24],主要由抗氧化物质AsA和GSH与AsA-GSH循环关键酶组成。研究[25]表明,植物体内AsA-GSH循环的快速运转可减少植物体内活性氧自由基含量,保持ROS平衡,缓解植物过氧化进程。AsA-GSH循环中活性最强的酶是APX和脱氢抗坏血酸还原酶,其活性的强弱直接影响AsA-GSH循环清除ROS的效率[26]。APX是AsA-GSH循环中主要的清除酶[27],而GSH的主要作用表现为促进膜蛋白结构稳定[28],同时GR活性可直接影响GSH含量[29]。本研究发现,干旱胁迫处理下ASA和GSH含量显著上升,这说明大豆体内的氧化应激机制在起作用。叶面喷施褪黑素后,ASA、GSH、GSSG和GR的含量相比干旱胁迫处理均提高,这表明褪黑素可以快速激活AsA-GSH循环,提高关键酶活性,维持AsA-GSH循环的高速运转并清除大豆体内的ROS副产物。王诗雅等[27]研究发现,逆境胁迫下,植物体内AsA、DHA和GSSG可保护细胞膜免受胁迫损伤。本试验结果表明,干旱胁迫导致大豆叶片AsA、DHA和GSSG含量增加,褪黑素处理则进一步提高了AsA、DHA和GSSG的含量,这与王诗雅等[27]的研究结果相同,说明褪黑素不但可以提升AsA-GSH循环中清除机制的效率,还可以增强AsA-GSH循环中的保护机制。
4 结论
干旱胁迫导致大豆叶片产生大量ROS,细胞膜脂过氧化程度加剧,褪黑素可提高干旱胁迫下大豆叶片AsA和GSH含量,增强APX和GR活性,促进干旱胁迫下大豆体内AsA-GSH循环的运转,减少干旱胁迫造成的膜脂过氧化损伤,进而提高大豆的抗旱性,减少干旱胁迫对产量的不利影响。
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鼓粒期是大豆碳氮代谢最复杂的阶段, 干旱胁迫必然限制鼓粒期大豆碳氮同化、分配和转移, 影响大豆产量的形成。在我们前期的研究中, 明确了外源褪黑素对干旱胁迫下鼓粒期大豆抗旱和碳氮代谢的生理调控效应。本研究通过转录组和代谢组分析来确定褪黑素对大豆干旱条件反应的一些重要的碳氮代谢基因和途径。转录组分析表明, 与干旱胁迫处理相比, 正常供水和干旱胁迫下喷施外源褪黑素处理的大豆叶片共同上调和下调的基因分别有37个和493个。上调的基因中存在着直接和间接参与碳氮代谢的功能基因, 包括正向调控的参与半胱氨酸合成、光合作用、碳水化合物代谢和葡萄糖代谢等途径关键基因。代谢组分析发现, 与干旱胁迫处理相比, 正常供水和干旱胁迫下喷施外源褪黑素处理的大豆叶片共同上调和下调的代谢物分别有17个和43个, 上调的代谢物中绝大部分(14/17)属于氨基酸、脂质、有机酸和碳水化合物, 进一步揭示了外源褪黑素能够提高大豆碳氮代谢与抗旱的能力。结合转录组和代谢组分析发现, 褪黑素通过调节氨基酸代谢和淀粉蔗糖代谢途径, 促进干旱胁迫下β-葡萄糖苷酶基因表达, 提高了L-天冬酰胺和6-磷酸葡萄糖代谢物的含量, 最终提高了大豆的抗旱性。
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