Journal of Plant Physiology 193(2016)57–63
Contents lists available at ScienceDirect
Journal of Plant
Physiology
一线口语j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j p l p
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Physiology
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Graphene oxide modulates root growth of Brassica napus L.and regulates ABA and IAA concentration
safari是什么Fan Cheng a ,1,Yu-Feng Liu a ,1,Guang-Yuan Lu b ,Xue-Kun Zhang b ,Ling-Li Xie a ,Cheng-Fei Yuan a ,Ben-Bo Xu a ,∗
a
College of Life Science,Yangtze University,Jingzhou 434025,China
b
Oil Crops Rearch Institute of the Chine Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture,Wuhan 430062,China
a r t i c l e
i n f o
Article history:
Received 14October 2015
Received in revid form 28January 2016Accepted 5February 2016
Available online 3March 2016
Keywords:ABA
Graphene oxide IAA
Root growth
Transcript abundance
a b s t r a c t
Rearchers have proven that nanomaterials have a significant effect on plant growth and develop-ment.To better understand the effects of nanomaterials on plants,Zhongshuang 11was treated with different concentrations of graphene oxide.The results indicated that 25–100mg/l graphene oxide treat-ment resulted in shorter minal root length compared with the control samples.The fresh root weight decread when treated with 50–100mg/l graphene oxide.The graphene oxide treatment had no signif-icant effect on the Malondialdehyde (MDA)content.Treatment with 50mg/l graphene oxide incread the transcript abundance of genes involved in ABA biosynthesis (NCED ,AAO,and ZEP )and some genes involved in IAA biosynthesis (ARF2,ARF8,IAA2,and IAA3),but inhibited the transcript levels of IAA4and IAA7.The graphene oxide treatment also resulted in a higher ABA content,but a lower IAA content com-pared with the control samples.The results indicated that graphene oxide modulated the root growth of Brassica napus L.and affected ABA and IAA biosynthesis and concentration.
©2016Elvier GmbH.All rights rerved.
1.Introduction
Since the discovery of graphene in 2004(Geim and Novolov,2007),increasing attention has been placed on exploring its physical properties (Service,2009).Nanotechnology has been increasingly applied to new materials,medicines,energy,electron-ics,and environmental protection (Kim et al.,2013;Nair et al.,2012;Sharma,2014;Zhang et al.,2012).The application of nanomate-rial bionsors in photo-electronics has incread in the last few years becau of their relatively high nsitivity (Germarie et al.,2009;Holzinger et al.,2009).Nanomaterials are currently success-fully applied in medicine and medical devices,and novel nano-drug delivery systems have been demonstrated to be more effective and convenient than common materials (Li et al.,2009;Sharma et al.,2009).如何提高口语表达能力
Abbreviations:AAO,abscisic acid aldehyde oxida;ABA,abscisic acid;ARF,auxin respon factor;IAA,indole-3-acetic acid;GA,gibberellin;MDA,malondi-aldehyde;NCED,9-cis -epoxycarotenoid dioxygena;ZEP,zeaxanthin epoxida.∗Corresponding author.Fax:+867168066257.
E-mail address:benboxu@yangtzeu.edu (B.-B.Xu).1
The authors contributed equally to this study.
However,limited rearch has been conducted on the applica-tion of graphene in agriculture.Considering that plants are primary producers in ecosystems,understanding the cross-talk between nanomaterials and plants is important to manipulate the effect of the nanomaterials on ecosystems.It is therefore esntial to under-stand the ecological risk and health significance of the graphene material before using it in agriculture.Many rearchers have demonstrated that nanomaterials can be relead into the environ-ment.However,some rearchers have reported the potential risks associated with the u of nanomaterials.Some reports have been published on the effects of nanomaterials in biology (Shen et al.,2009;Xue et al.,2000),while others have proven that nanoparti-cles can potentially enter cells and accumulate in tissues,resulting in damaged cells and tissue lesions (Liu et al.,2009;Tang et al.,2009).
The application of nanomaterials in agriculture started in 2007and since then,much attention has been paid to this field (Lin and Xing,2007;Liu et al.,2015;Mushtaq,2011).For example,rearchers (Khodakovskaya et al.,2009)reported that nanotubes penetrated the husk of tomato eds and greatly incread the rate of ed germination.Engineered nanomaterials are constantly being deployed in agricultural fields with unknown impacts on crop species (Bandyopadhyay et al.,2013;St
ampoulis et al.,2009).Lin
dx.doi/10.1016/j.jplph.2016.02.011
0176-1617/©2016Elvier GmbH.All rights rerved.
58 F.Cheng et al./Journal of Plant Physiology193(2016)57–63
Table1
Primers for real time PCR.
Gene name Primers Sequence Annealing temperature(◦C)
ACT ACTF5 -CTGGAATTGCTGACCGTATGAG-3 58
ACTR5 -ATCTGTTGGAAAGTGCTGAGGG-3
NCED NCEDF5 -GTCGCGTCAACCTCCAAGCT-3 56
NCEDR5 -TCTGTTTCTCCCCGGAGAGG-3
AAO AAOF5 -CAACAGTGGACACCACAAGAAC-3 56
AAOR5 -CAACAGTGGACACCACAAGAAC-3等待的英文
ZEP ZEPF5 -CAACAGTGGACACCACAAGAAC-3 56
ZEPR5 -CAACAGTGGACACCACAAGAAC-3
IAA2IAA2F5 -CAACAGTGGACACCACAAGAAC-3 56
IAA2R5 -CAACAGTGGACACCACAAGAAC-3
IAA3IAA3F5 -CAACAGTGGACACCACAAGAAC-3 56
IAA3R5 -CAACAGTGGACACCACAAGAAC-3
IAA4IAA4F5 -CAACAGTGGACACCACAAGAAC-3 56
IAA4R5 -CAACAGTGGACACCACAAGAAC-3
IAA7IAA7F5 -CAACAGTGGACACCACAAGAAC-3 56
IAA7R5 -CAACAGTGGACACCACAAGAAC-3谦冲
ARF2ARF2F5 -CAACAGTGGACACCACAAGAAC-3 56
ARF2R5 -CAACAGTGGACACCACAAGAAC-3
ARF8ARF8F5 -CAACAGTGGACACCACAAGAAC-3 55
ARF8R5 -CAACAGTGGACACCACAAGAAC-3
and Xing(2007)found that nanoparticles significantly inhibited ed germination and root growth.Graphene oxide was also found to decrea biomass and root number and increa oxidative stress, all thought to be regulated by its metabolism(Hu et al.,2014).
To better understand the effects of graphene on plants,rape edlings were ud as study materials in this study.The rape edlings were treated with different concentrations of graphene and as a result,we found that25–100mg/l of graphene had a sig-nificant effect on root growth.Thefindings motivated us to study the underlying mechanisms.
2.Materials and methods
2.1.Materials
All experiments were performed on cultivated“Zhongshuang 11”from the Oil Crops Rearch Institute of the Chine Academy of Agricultural Sciences.Rapeeds were germinated on wet sterilized filter paper in80-cm petri dishes in a growth chamber at25±1◦C, with a photoperiod of24h in the dark.
2.2.Effects of graphene on the growth of rape roots
Seedlings(8days old),with uniform growth,were chon for the experiments.The edlings were placed on sterilized sponges in a plastic bowl in a growth chamber at25±1◦C,with a photope-riod of16h of light and8h of dark.The sponges were soaked in distilled water or graphene oxide to expo the edlings to differ-ent stress treatments(0,5,10,25,50and100mg/l graphene oxide). The edlings were harvested for measurements after treatment.
2018年6月英语四级答案2.3.Determination of root length and fresh weight
At least three edlings were randomly lected from tho that received the graphene oxide treatment for15days.Maximum root length was measured using a ruler,and root length(cm)was defined as the length from the root tip to the ba of root.
The edling roots were cut at the ba and weighed to deter-mine the fresh root weight.
2.4.Determination of the MDA content
MDA was measured by reaction with2-thiobarbituric acid according to a previously reported method(Cakmak and Marschner,1992).Briefly,0.3g of fresh material from each edling was homogenized in3ml of50mM PBS(pH7.8)containing0.2M EDTA and the homogenate was centrifuged at12,000×g for20min at4◦C.Then,0.5ml of supernatant was added to2ml of10%(w/v) TCA and2ml of0.5%(w/v)TBA(2-thiobarbituric acid).The mix-ture was heated in a water bath shaker at95◦C for30min and then rapidly cooled in an ice-bath.The absorbance was measured at532nm after centrifugation at5000×g for10min,and the value for non-specific absorption at600nm was measured using a spec-trophotometer.The concentration of MDA was calculated from the absorbance at532nm(correction was performed for unspecific turbidity by subtracting the absorbance at600nm).
2.5.Determination of transcript abundance
Total RNA was extracted from edling or root samples using Tri-zol(Invitrogen,US)and treated with RNa-free DNa I(Sangon Biotech,China)to remove contaminating genomic DNA.First-strand complementary DNA(cDNA)was synthesized according to the Superscript Rever Transcripta ma
nual(Invitrogen,US).
Quantitative real-time PCR was performed with a7300 Real-Time PCR System(Applied Biosystems)using SYBR Green SuperReal qPCR PreMix(Tiangen,China).The-Actin gene was ud as the internal control to normalize the sample variance.Relative quantification of the transcript abundance of each gene was per-formed using the Livak method(Livak and Schmittgen,2001).The primers ud for determining transcript abundance are listed in Table1.
2.6.IAA and ABA quantification
A0.1-g sample was ground using a chilled mortar and liquid N2, and transferred to a tip containing1.2ml cold80%methanol and 10mg/l butylated hydroxytoluene.The pellet was incubated in ice for30min,centrifuged for5min at1000×g and the supernatant was collected.The pellet was extracted twice with0.5ml cold80% methanol and10mg/l butylated hydroxytoluene.All of the super-natant was transferred to a4ppak C18column(Millipore Waters, USA)and eluted with80%methanol.All elute was collected and evaporated under vacuum at45◦C.The dry sample was analyzed according to the manuals of the Phytodeted IAA Test Kit and the Phytodeted ABA Test Kit(Agdia,France).
F.Cheng et al./Journal of Plant Physiology193(2016)57–63
59
油泵选型
Fig.1.Effect of graphene oxide on root morphology(A)and leaves morphology(B).
2.7.Statistical analysis of data
DPS7.05software was ud for significance testing and Excel 2007was ud to construct the tables.The values are reported as the means and their standard errors.Differences were considered significant at P<0.05or P<0.01.
3.Results
3.1.Effect of graphene oxide on rape root growth and
development
To investigate the effect of graphene oxide on the growth and development of roots of B.napus,the edlings were treated with0–100mg/l of graphene oxide.After10days treatment,the results indicated that the lower concentration of graphene oxide (<25mg/l)had no significant effect on root length,but the higher concentration(≥25mg/l)inhibited root growth(Fig.1).The results also indicated that adventiti
ous roots were inhibited by graphene oxide treatment.There were numerous adventitious roots in the control samples,but the number of adventitious roots decread as the concentration of graphene oxide incread.Similar results were obrved after15days treatment.The average minal root length of the control samples was23.11cm after15days treat-ment.Treatment with5–10mg/l graphene oxide had no significant effect on root length,but a graphene oxide concentration higher than25mg/l significantly decread root length(Fig.2).A greater inhibitory effect was obrved as the graphene oxide concentration incread from25–100mg/l.When the treatment concentration was100mg/l,the root length was only10.38cm.The edlings were transplanted into soil,and the plant morphology,leaves,and stems were examined,but no significant differences were
found Fig.2.Effect of different concentration graphene oxide treatments on root length of B.napus after15days treatment.Values with different letters were significantly different(Student’s t-test,P<
0.01).
Fig.3.Effect of different concentration graphene oxide on root fresh weight of B.napus after15days treatments.Values with different letters were significantly different(Student’s t-test,P<0.01).
between the treated and control samples except that the control samples were thinner(Fig.1).
The fresh root weights of the treated samples were also mea-sured.The average fresh root weight of the control samples was 0.35mg after15days treatment.Treatment with a low concentra-tion(5–25mg/l)of graphene oxide had no significant effect on the fresh root weight of B.napus(Fig.3).At a graphene oxide concentra-tion of50mg/l,the fresh root weight was strongly reduced,and at a graphene oxide concentration of100mg/l,the fresh root weight was only0.2g.
3.2.Effect of graphene oxide on the MDA content
Lipid peroxidation in edlings was measured as the content of MDA(Fig.4).The results indicated that treatment with5–100mg/l of graphene oxide had no significant effect on the MDA content, which indicated that the MDA content was not the key reason for the changes obrved in root length and fresh weight.
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F.Cheng et al./Journal of Plant Physiology 193(2016)
57–63
Fig.4.Effects of graphene oxide on MDA content in B.napus .Values with different letters were significantly different.(Student’s t-test,P <
0.01).
Fig.5.Effects of graphene oxide on transcript abundance of key genes for ABA and IAA biosynthesis.Different letters indicate significant differences between 0and 50mg/l graphene oxide treatment (Student’s t-test,P <0.05).Results are the means of three biological replicates.
3.3.Effect of graphene oxide on the transcriptional level of key genes for ABA biosynthesis
Several hormonal pathways are involved in the regulation of root growth,especially the auxin and cytokinin pathways.9-cis-epoxycarotenoid dioxygena (NCED ),Abscisic acid aldehyde oxida (AAO ),and Zeaxanthin epoxida (ZEP )are three key genes for ABA biosynthesis.To understand the effect of graphene oxide on ABA biosynthesis and root growth,the transcriptional abundance of NCED ,AAO,and ZEP was detected using real-time PCR.The results indicated that the 50mg/l graphene oxide treatment incread the transcript abundance of NCED ,AAO and ZEP (Fig.5).Treatment with 50mg/l of graphene oxide incread the relative transcript abundance of NCED approximately 4.3-fold higher than that mea-sured in the control,and the transcript abundance of AAO and ZEP after treatment was 2.8-and 1.3-fold higher,respectively,than that measured in the control samples.Becau NCED is a rate-limiting enzyme for ABA biosynthesis,the increa in the NCED transcrip-tional level may have resulted in the higher ABA content in the graphene oxide-treated
relaxed
短篇英语作文samples.
Fig.6.Effect of graphene oxide treatment on ABA content.Different letters indicate significant differences between 0and 50mg/l graphene oxide treatments (Student’s t-test,P <0.01).Results are the means of three biological replicates.
3.4.Effect of graphene oxide on the transcriptional level of key genes for IAA biosynthesis
In plants,auxin regulates the transcriptional level of many auxin-responsive genes through auxin/indole-3-acetic acid (Aux/IAA)–AUXIN RESPONSE FACTOR (ARF)auxin-signaling mod-ules (De Smet et al.,2010).There are 22ARFs and 29Aux/IAA proteins in Arabidopsis (Goh et al.,2012;Guilfoyle and Hagen,2007).The relative transcript abundance of six reprentative ARFs or Aux/IAA genes was detected using real-time PCR.The results indicated that the 50mg/l graphene oxide treatment incread the transcript abundance of ARF2,ARF8,IAA2,and IAA3,but decread the transcript abundance of IAA4and IAA7.
3.5.Graphene oxide regulates the ABA content
The real-time PCR results indicated that graphene oxide induced the expression of some key genes
involved in ABA biosynthesis.Conquently,the ABA content was measured.The ABA con-tent in the control and 5treated samples (5,10,25,50,and 100mg/l graphene oxide treatment)was 69.10,82.33,103.89,364.37,372.91,and 401.97ng g −1FW,respectively.The ABA con-tent was significantly incread by treatment with 5–100mg/l of graphene oxide (Fig.6).The ABA content in the treated samples incread with an increament in the concentration of graphene oxide.The ABA content in the 100mg/l treated sample was 5.8-fold higher than that measured in the control sample.
3.6.Graphene oxide regulates the IAA content
The IAA content in the control sample and 5treated samples (10,15,25,50and 100mg/l)was 40.23,41.17,37.30,30.93,26.97,and 26.31ng g −1FW,respectively.Treatment with 10–100mg/l of graphene oxide significantly decread the IAA content.The IAA content decread with an increament in the graphene oxide concentration.The IAA content in the 100mg/l treated sample was 26.34ng g FW,which was lower than that measured in the control sample (Fig.7).
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Fig.7.Effect of graphene oxide treatment on IAA content.Different letters indicate significant differences between 0and 50mg/l graphene oxide treatment (Student’s t-test,P <0.01).Results are the means of three biological replicates.
4.Discussion
4.1.Graphene oxide significantly affected plant root growth
Investigations have shown that nanomaterials are able to pro-duce stress in plants,resulting in an excess production of reactive oxygen species (ROS)that have the potential to affect proteins,lipids,carbohydrates,and DNA (Liu et al.,2013a;Liu et al.,2010).Our results indicated that the graphene oxide treatment had no significant effect on the MDA content of B.napus ,but it did affect the SOD,POD and CAT enzyme activities (Fig.8).SOD,POD and CAT enzyme activity usually increas under stress caud by saline conditions,drought,or wounding.Transcriptome anal-ysis showed similar expression patterns for Arabidopsis plants expod to nanoparticles and other stress inducers (saline,drought,or wounding)(Garcia-Sanchez et al.,2015).This indicates that nanoparticles are a type of stress for plants.
The results indicated that treatment with 25–100mg/l of graphene oxide significantly inhibited the roo
t growth of B.napus .Other rearchers have also found that a high concentration of graphene (>50mg/l)suppresd root growth in rice (Liu et al.,2015),and incread the root length of tomato (Zhang et al.,2015).Rearchers also (Tripath and Sarkar,2015)reported that carbon
nanotubes enhanced root and shoot growth under light and dark conditions.However,the inhibitory effect of carbon nanotubes on plant growth has been reported by many rearchers (Begum et al.,2014).Graphene quantum dots enhanced the growth rate of corian-der and garlic plants,including the leaves,roots,shoots,flowers,and fruits (Chakravarty et al.,2015).Other experiments indicated that different types of nanoparticles had positive or negative effects on plant growth (Khodakovskaya et al.,2009;Ma et al.,2010).The experiments indicate that the effects of nanomaterials on plant growth and development are complicated and depend on the type of nanoparticle,plant genotype,experimental conditions and phytohormone concentration of plants.
4.2.Graphene oxide could affect the IAA pathway in plants
Root elongation is an important process for plant growth and development.Primary root elongation is an auxin-associated phenotype.Auxin activates H +-ATPa in plants through phospho-rylation to acidify the cell wall and enhance the activity of the cell wall,which promotes root elongation during the early pha of hypocotyl elongation (Takahashi et al.,2012).
Becau the level of auxin in plants is optimal under normal conditions,root elongation is highly nsitive to variations in the concentration of auxin.Auxin levels are strongly regulated through auxin biosynthesis,storage and transport,which regulate root elon-gation.
Exogenous auxin positively or negatively affects root growth depending on the concentration and ratios of endogenous hor-mones.A low concentration may promote root growth and elongation,but higher concentrations may inhibit growth.Mutant yucca has longer hypocotyls and shorter primary roots than the wild type becau of higher levels of IAA (Zhao et al.,2001).Indole-3-acetic acid (IAA)is the most characterized molecular signal for root system architecture and growth.Auxin can stimulate root growth by activating quiescent pericycle cells to initiate division (Ivanchenko et al.,2010).Appropriate synthesis,signaling and transport of auxin are required for root formation (Peret et al.,2009).
The 50mg/l graphene oxide treatment incread the transcript abundance of ARF2,ARF8,IAA2and IAA3,but lowered the transcript abundance of IAA4and IAA7,which are important genes for IAA syn-thesis.This indicates that graphene oxide can affect IAA synthesis and regulate plant growth.
A low concentration of graphene oxide (10mg/l)slightly decread the IAA content (37.3ng g FW)compared with the con-trol (40.2ng g FW)and had no significant effect on root
length.
Fig.8.Effects of graphene oxide on activities of SOD (A),CAT (B)and POD (C)in B.napus .At each treatment,values with different letters are significantly different at P <0.05or P <0.01.
