丙烯酰胺/羧甲基纤维素/生物炭复合水凝胶对烟苗镉胁迫的缓解效应研究
Effective Effects of Acrylamide/Carboxymethyl Cellulose/Biochar Composite Hydrogel on Cadmium Stress in Tobacco Seedlings
通讯作者:
第一联系人: 夏茂林为共同第一作者,主要从事烟草化学研究,E-mail:
收稿日期: 2021-07-15 修回日期: 2021-09-26 网络出版日期: 2022-05-07
基金资助: |
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Received: 2021-07-15 Revised: 2021-09-26 Online: 2022-05-07
作者简介 About authors
杜甫,主要从事烟草栽培及烟草化学研究,E-mail:
为缓解镉(Cd)对烟苗生长发育的毒害作用,考察新型丙烯酰胺/羧甲基纤维素/生物炭复合水凝胶(AM/CMC/B)对水溶液中Cd2+的去除效果,并通过盆栽试验探讨AM/CMC/B缓解烟苗Cd胁迫的效应。结果表明,AM/CMC/B可有效去除水中Cd2+,改善Cd胁迫下烟苗表型;AM/CMC/B处理下烟苗的最大叶长、最大叶宽、最大叶面积、总根长、总根表面积、平均根径、根尖数和根体积分别显著增加38.28%、30.01%、79.19%、83.31%、83.97%、54.44%、88.21%和261.56%;烟苗地上部分的鲜重和干重分别增加了98.39%和84.22%,地下部分的鲜重和干重分别增加了130.67%和231.82%,烟苗净光合速率、SPAD值和气孔导度显著增加62.03%、29.61%和84.42%,丙二醛含量显著降低36.61%,烟苗抗氧化活性和代谢物含量提高。可见,AM/CMC/B可以显著提高Cd胁迫下烟苗的耐受性。
关键词:
In order to alleviate the toxic effect of cadmium (Cd) on the growth and development of tobacco seedlings, the removal effects of new acrylamide/carboxymethylcellulose/biochar composite hydrogel (AM/CMC/B) on Cd2+ in aqueous solution was investigated, and the effects of AM/CMC/B on alleviating Cd stress of tobacco seedlings was studied by pot experiment. The results showed that AM/CMC/B could effectively remove Cd2+ from water and improve the phenotype of tobacco seedlings under Cd stress. Under AM/CMC/B treatment, the maximum leaf length, maximum leaf width, maximum leaf area, total root length, total root surface area, average root diameter, root tip number and root volume increased significantly by 38.28%, 30.01%, 79.19%, 83.31%, 83.97%, 54.44%, 88.21% and 261.56%, respectively. The fresh and dry weight of aboveground part of tobacco seedlings increased by 98.39% and 84.22%, the fresh and dry weight of underground part of tobacco seedlings increased by 130.67% and 231.82%, respectively. The photosynthetic rate, SPAD value and stomatal conductance of tobacco seedlings significantly increased by 62.03%, 29.61% and 84.42%, the content of malondialdehyde decreased by 36.61%. The antioxidant activity and metabolite content of tobacco seedlings increased. AM/CMC/B could significantly improve the tolerance of tobacco seedlings for Cd stress.
Keywords:
本文引用格式
杜甫, 夏茂林, 刘新源, 于兆锦, 张展, 刘云飞, 姬小明.
Du Fu, Xia Maolin, Liu Xinyuan, Yu Zhaojin, Zhang Zhan, Liu Yunfei, Ji Xiaoming.
1 材料与方法
1.1 水凝胶的制备及表征
1.1.1 化学试剂
羧甲基纤维素钠(CMC,聚合度为800~1200),丙烯酰胺(AM)、过硫酸铵(APS)和N,N'-亚甲基双丙烯酰胺(MBA)购自天津市科密欧化学试剂有限公司,Cd(NO3)2·4H2O(上海伊恩试剂有限公司),三氟乙酰胺(BSTFA)、N,N'-二甲基甲酰胺(DMF)和CH2Cl2由天津大茂化学试剂厂生产。上述试剂均为分析纯,试验用水均为去离子水。花生壳生物炭(B)由河南惠农土质保育有限公司提供(450℃,灼烧4h)。
1.1.2 水凝胶AM/CMC的制备
将CMC 1.0g置于100mL烧杯中并加入100mL去离子水,搅拌直至CMC完全溶解。然后以8:1(AM:CMC)的质量比加入AM 8.0g。之后加入APS 0.09g和MBA 0.045g,并用玻璃棒搅拌均匀,调节磁力搅拌器转速为200转/min,水浴温度为60℃,反应3h,得水凝胶AM/CMC。
1.1.3 水凝胶AM/CMC/B的制备
将CMC 1.0g和100mL去离子水置于100mL烧杯中,搅拌直至CMC完全溶解。然后将AM 8.0g和B 4.0g加入烧杯中。之后加入APS 0.09g和MBA 0.045g,并用玻璃棒搅拌均匀,调节磁力搅拌器转速为200转/min,水浴温度为60℃,反应3h,得水凝胶AM/CMC/B。
将制备的水凝胶AM/CMC和AM/CMC/B在去离子水中吸水24h使其充分溶胀,然后用去离子水冲洗不小于3次。再将其置于60℃真空干燥箱中干燥至恒重,利用粉碎机粉碎,用0.3mm(60目)孔径筛过滤,密封保存于真空干燥器中。
1.1.4 水凝胶的表征
将2种水凝胶样品与KBr混合压片,在4000~500/cm的条件下,利用Nicolet iS10型傅里叶变换红外光谱仪测定2种水凝胶的FT-IR图谱。
1.1.5 水凝胶对Cd2+的吸附性能研究
分别取AM/CMC和AM/CMC/B各0.05g干凝胶加入到100mL Cd2+溶液中。研究固定温度(25℃)、干凝胶量(0.05g)、固定初始Cd2+浓度(200mg/L)和不同pH(2~6)条件下,2种水凝胶对Cd2+的平衡吸附量(Qe)。吸附试验在100mL塑料瓶中进行,调节振荡器速度为150转/min。达到平衡后,通过Vista-MPX ICP-OES电感耦合等离子体原子发射光谱仪测定Cd2+的浓度。最后,2种水凝胶的最大吸附容量(Qm)通过以下等式计算:
式中,C0和Ce分别是溶液中Cd2+初始浓度和最终浓度(mg/L)。V和m分别是Cd2+溶液的体积(L)和2种干燥凝胶的质量(g)。
1.2 试验设计
土壤取自河南农业大学许昌校区(总Cd 0.13mg/kg,pH 7.6,有机质19.09g/kg,碱解氮74.70mg/kg,有效磷8.70mg/kg,速效钾114.50mg/kg,水溶性氯24.80mg/kg)。将拥有4片真叶的烟苗移栽至塑料盆内,塑料盆的尺寸为7cm×7cm(直径×高),每盆装风干土200g。试验设置3个处理,CK(空白对照,不添加任何水凝胶)、H(每盆添加干凝胶AM/CMC 0.50g)、BH(每盆添加干凝胶AM/CMC/B 0.50g),每个处理的10株烟苗作为重复。移栽前,将干凝胶和干土混合均匀,然后将10mg/kg的Cd2+溶液等量加入到每个盆中。为防止Cd2+渗出,每个盆底部用胶带密封。将移栽后的烟苗置于恒温培养箱中培养,温度为25℃±1℃,每天光照16h。培养15d后对烟苗进行拍照和指标测定,同时取烟叶样品用液氮冷冻后储存于-80℃冰箱中,进行后续分析。
1.3 测定项目与方法
1.3.1 烟苗表型记录及生长指标
采用尼康D610相机分别于Cd胁迫0和15d对烟苗表型进行拍照。用EPSON Perfectio V800 Photo根系扫描仪(日本EPSON公司)扫描烟苗根系获得根系图片,利用根系分析软件WinRHIZO对烟苗根系生长指标进行分析。手工测定烟苗的最大叶长、最大叶宽和最大叶面积。
1.3.2 生物量和相对含水量(RWC)
在Cd胁迫15d后,采用文献[15]的方法测定烟苗的生物量,并利用公式计算新鲜烟叶的RWC:
式中,FW(g)为新鲜烟叶饱和吸水后的质量,TW(g)为新鲜烟叶的质量,DW(g)为新鲜烟叶的干重。
1.3.3 光合作用和相对叶绿素含量(SPAD值)
参照文献[16]方法,采用LI-6400型光合仪(美国LI-COR公司)测定烟苗叶片净光合速率(Pn)、胞间CO2浓度(Ci)、蒸腾速率(Tr)和气孔导度(Gs)。采用SPAD 502型便携式叶绿素仪测定SPAD值。
1.3.4 丙二醛(MDA)含量和抗氧化酶活性
参照文献[17]的方法测定叶片中MDA含量,并采用北京索莱宝科技有限公司生产的试剂盒测定叶片脯氨酸(Pro)含量、过氧化氢酶(CAT)和过氧化物酶(POD)活性。
1.3.5 代谢物种类和含量
称量冷冻干燥的烟草粉末20mg于2mL离心管中,然后加入内标萃取物1.5mL(1L CH2Cl2含有2.5mg十三烷基酸),在室温下搅拌均匀,然后超声60min。经氮气吹干后,加入衍生化试剂(BSTFA/DMF,体积比1:1),采用美国安捷仑7890B-5977A型气质联用仪测定烟草代谢物的种类和含量。
1.4 数据处理
采用DPS 7.0软件进行差异性分析,用Graph Pad Prism 8.0和AI 2017软件绘制图表。
2 结果与分析
2.1 水凝胶FT-IR分析
图1
图1
AM/CMC/B(a)、AM/CMC(b)和B(c)的FT-IR
Fig.1
FT-IR of AM/CMC/B(a), AM/CMC(b) and B(c)
2.2 pH对水凝胶Cd2+平衡吸附量的影响
由图2可知,AM/CMC/B在pH为2~6的Cd2+溶液中的平衡吸附量呈现出先增加再减小的趋势,在pH为2和5时,平衡吸附量分别达到最小值和最大值,分别为97.53和164.83mg/g。当pH为2时,AM/CMC/B与AM/CMC的平衡吸附量相差最小,为11.27mg/g;当pH为4时,AM/CMC/B与AM/CMC的平衡吸附量相差最大,为56.56mg/g。可见,在添加了生物炭形成水凝胶后,对溶液中Cd2+的吸附量大大增加,可能与生物炭对Cd2+有良好的吸附性有关。
图2
图2
不同水凝胶在不同pH下对Cd2+的平衡吸附量
Fig.2
Equilibrium adsorption of Cd2+ by different hydrogels under different pH values
2.3 水凝胶对烟苗表型和生长指标的影响
由图3可知,与CK处理相比,H和BH处理的烟苗叶片数增加1片,叶片较大、颜色较深、根系更发达,说明H和BH处理可以缓解镉胁迫对烟苗生长发育的限制,改善烟苗表型。由图4可知,与CK处理相比,H和BH处理的最大叶长分别增加了14.94%和38.28%,最大叶宽分别增加了1.94%和30.01%,最大叶面积分别增加了16.90%和79.19%,总根长分别增加了29.74%和83.31%。总根表面积分别增加了33.00%和83.97%,根平均直径分别增加12.36%和54.44%,根尖数分别增加了14.83%和88.21%,根体积分别增加了69.75%和261.56%。这些数据表明,Cd对不加任何水凝胶处理的烟苗毒害最大,使其生长发育受限,H和BH处理均可减轻Cd胁迫下烟苗受到的毒害效应,以BH处理的改善效果较好。
图3
图3
Cd胁迫15d烟苗的表型
Fig.3
Phenotype of tobacco seedlings under Cd stress for 15 days
图4
图4
Cd胁迫15d烟苗的生长指标
不同小写字母表示在0.05水平上差异显著,下同
Fig.4
Growth indexes of tobacco seedlings under Cd stress for 15 days
Different lowercase letters indicate significant difference at 0.05 level, the same below
2.4 水凝胶对烟苗生物量和RWC的影响
由图5可知,H和BH处理烟苗的地上部鲜重、地下部干重和叶片RWC均显著高于对照。在Cd胁迫下,CK处理烟苗受Cd影响最大。与CK处理相比,H和BH处理的地上部鲜重分别增加25.91%和98.39%,地下部鲜重分别增加了20.27%和130.67%,地上部干重分别增加了18.10%和84.22%,地下部干重分别增加了68.18%和231.82%。叶片RWC分别提高了4.84%和17.01%。可见,AM/CMC和AM/CMC/B水凝胶能够缓解Cd胁迫下烟苗的毒害,提高烟苗的抗逆性。AM/CMC/B水凝胶对提高Cd胁迫下烟苗的生物量和RWC的效果较好。
图5
图5
Cd胁迫15d烟苗的生物量和叶片RWC
Fig.5
Biomass and leaf RWC of tobacco seedlings under Cd stress for 15 days
2.5 水凝胶对烟苗光合特性和SPAD值的影响
从图6可知,与CK处理相比,H处理烟苗的Pn、Ci、SPAD值、Tr和Gs分别提高了28.47%、29.67%、11.28%、43.20%、52.63%,BH处理分别提高了62.03%、31.59%、29.61%、82.54%、84.42%。H和BH处理能提高Cd胁迫下烟苗的SPAD值和光合作用。总之,在Cd胁迫15d后,BH处理的烟草幼苗光合作用受到的抑制最小。可以推测,BH处理的光合活性增强可能归因于其土壤中的Cd被有效吸附和固定。
图6
图6
Cd胁迫15d烟苗的光合特性和SPAD值
Fig.6
Photosynthetic characteristics and SPAD values of tobacco seedlings under Cd stress for 15 days
2.6 水凝胶对烟苗MDA和Pro含量及CAT和POD活性的影响
由图7可知,与CK处理相比,BH处理显著提高了Pro含量及CAT和POD活性,显著降低了MDA含量,H处理显著提高了CAT和POD活性。与CK相比,H处理烟苗的Pro含量及CAT和POD活性分别提高了57.53%、95.32%、67.10%,BH处理分别提高了192.19%、226.93%、105.77%,H和BH处理MDA含量分别降低了17.30%和36.61%。可能因为AM/CMC和AM/CMC/B通过增强烟苗抗氧化能力,明显降低Cd诱导的氧化损伤水平。此外,AM/CMC和AM/CMC/B均提高了叶片抗氧化酶活性,其中AM/CMC/B水凝胶的效果较好。
图7
图7
Cd胁迫15d烟苗的MDA和Pro含量及CAT和POD活性
Fig.7
Contents of MDA and Pro, activities of CAT and POD in tobacco seedlings under Cd stress for 15 days
2.7 水凝胶对烟苗代谢物含量的影响
GC-MS分析结果(表1)显示,从烟叶中共检测到30种烟草代谢物,包括有机酸(7种)、萜类(8种)、烷烃(10种)和其他代谢物(5种)。4种代谢物总含量以BH处理最高,H处理次之,CK处理最低。CK处理中其他代谢物含量最高,烷烃类含量最低。H和BH处理中含量最高的是萜类,最低的是烷烃类。代谢物总量以BH处理的含量最高,CK处理最低。H和BH处理分别比CK处理高62.38%和123.13%,表明水凝胶AM/CMC和AM/CMC/B可提高Cd胁迫下烟叶代谢水平,从而提高烟苗对Cd的耐受性,减轻Cd的毒害作用,代谢物水平高有利于烟苗自身的生长发育,对外界逆境胁迫的抗性也有所增加,其中BH处理的效果较好。
表1 不同处理烟苗代谢物的含量
Table 1
代谢物类别 Metabolite category | 代谢物名称 Metabolite name | 保留时间 Retention time (min) | 含量Content (mg/g) | ||
---|---|---|---|---|---|
CK | H | BH | |||
有机酸Organic acid | 苹果酸 | 5.26 | 0.255 | 0.349 | 0.359 |
对苯二甲酸 | 6.91 | 0.151 | 0.206 | 1.407 | |
二十碳五烯酸 | 8.58 | 0.000 | 3.115 | 0.226 | |
棕榈酸 | 8.79 | 1.353 | 1.871 | 2.586 | |
亚油酸 | 10.80 | 0.239 | 0.380 | 0.792 | |
亚麻酸 | 10.91 | 1.288 | 2.221 | 5.113 | |
硬脂酸 | 11.26 | 0.547 | 0.729 | 0.993 | |
有机酸总量 | 3.833 | 8.872 | 11.476 | ||
萜类Terpenoids | 植醇 | 7.51 | 0.438 | 0.610 | 1.005 |
α-西柏三烯二醇 | 11.37 | 1.838 | 3.696 | 4.470 | |
β-西柏三烯二醇 | 12.28 | 0.851 | 1.032 | 1.274 | |
菜油甾醇 | 23.86 | 0.433 | 0.860 | 1.574 | |
豆甾醇 | 24.07 | 1.563 | 2.192 | 3.504 | |
β-谷甾醇 | 24.47 | 0.578 | 1.147 | 1.763 | |
β-香树脂醇 | 24.59 | 0.297 | 0.540 | 0.904 | |
鹅去氧胆酸 | 24.96 | 0.000 | 0.000 | 0.196 | |
萜类总量 | 5.998 | 10.076 | 14.691 | ||
烷烃类Alkanes | 烷烃Ⅰ | 21.34 | 0.000 | 0.136 | 0.258 |
烷烃Ⅱ | 21.60 | 0.000 | 0.133 | 0.197 | |
烷烃Ⅲ | 22.07 | 0.203 | 0.358 | 0.729 | |
烷烃Ⅳ | 22.22 | 0.102 | 0.155 | 0.301 | |
烷烃Ⅴ | 22.59 | 0.375 | 0.688 | 1.314 | |
烷烃Ⅵ | 22.79 | 0.689 | 0.902 | 1.538 | |
烷烃Ⅶ | 22.99 | 0.210 | 0.440 | 0.519 | |
烷烃Ⅷ | 23.13 | 0.121 | 0.329 | 0.461 | |
烷烃Ⅸ | 23.72 | 0.258 | 0.597 | 1.163 | |
烷烃Ⅹ | 23.96 | 0.267 | 0.813 | 1.240 | |
烷烃总量 | 2.225 | 4.550 | 7.719 | ||
其他代谢物Other metabolites | 丁基化羟基甲苯 | 6.51 | 0.134 | 0.203 | 0.258 |
新植二烯 | 7.23 | 3.753 | 3.423 | 3.214 | |
十八醇 | 10.00 | 0.000 | 0.000 | 0.128 | |
4,4'-亚甲基双(2-叔丁基-6-甲基苯酚) | 17.82 | 3.076 | 3.610 | 4.691 | |
单硬脂酸甘油酯 | 20.90 | 0.354 | 0.725 | 1.049 | |
其他代谢物总量 | 7.317 | 7.961 | 9.341 | ||
代谢物总量Total metabolites | 19.373 | 31.458 | 43.227 |
3 讨论
氧化应激是非生物和生物应激的核心部分。这种机制是由于细胞产生活性氧(ROS,包括H2O2和O2-)和抗氧化酶之间的严重失衡所致,从而导致严重的生理紊乱。酶促活性氧清除系统在维持膜的结构和功能以及细胞氧化还原平衡方面起着重要作用[24]。例如,Pro可以增强抗氧化反应,最终缓解Cd的抑制效应[25]。此外,氧化损伤与抗氧化防御机制密切相关。超氧化物歧化酶是抑制ROS产生的第一步,将O2-转化为H2O2。此外,H2O2可以被CAT分解成O2-和H2O,从而降低ROS的毒性[26]。Cd还会干扰植物体内的细胞酶系统和氧化应激反应。本试验中,添加水凝胶AM/CMC/B处理的烟苗MDA积累量明显降低,Pro含量和CAT、POD活性显著提升,表明AM/CMC/B提高了烟苗对体内ROS的清除能力,降低了Cd对细胞的毒害作用,从而有利于自身生长。
代谢物是植物体内重要的化合物,主要负责调节植物的生命活动和抵抗逆境。有机酸参与光合作用和呼吸作用,还可作为活性代谢产物调节渗透压,平衡过量阳离子。另外,有机酸对植物体内的重金属有解毒作用。萜类化合物也是植物体内重要的代谢产物,有些萜类化合物是植物生长发育所必需的,有些则在调节植物与环境的关系中起着重要的生态作用。植物中的烷烃在叶片表面形成一层蜡,为维持内部环境提供了保障,增强了叶片的抗逆性。Cd通过抑制植物代谢产物的生物合成来抑制植物生长[27]。本试验的结果表明,添加水凝胶处理H和BH将烟苗的代谢物总量分别提高了62.38%和123.13%,可见,AM/CMC/B对烟苗抵御Cd胁迫的效果最佳,降低了Cd对烟苗代谢产物生物合成所造成的抑制效果,促进其生长。
4 结论
本研究成功制备了丙烯酰胺/羧甲基纤维素/生物炭复合水凝胶AM/CMC/B,通过表征和吸附试验,表明其可以有效吸附水中的重金属Cd。盆栽试验说明其能够降低Cd在烟苗体内的积累,减少Cd的毒性,提高烟苗的抗氧化能力,促进生长发育,显著增加了代谢物含量,从而有效提高Cd胁迫下烟苗的耐受性,是一种理想的水体重金属污染吸附剂和土壤重金属污染改良剂。
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