Expression Analysis of 14-3-3 Gene in Tall Fescue under Several Abiotic Stresses

Xiaodong LI,Erru YU,Jianhong SHU,Jiahai WU,Xiaoli WANG*

1.Guizhou Institute of Prataculture,Guiyang 550006,China;2.Guizhou Institute of Oil Crops,Guiyang 550006,China

In the nature,plants sense and adapt to changes in environmental factors by regulating the expression of their own genes.Researchers have studied the effects of light,temperature,moisture,salinity and nutrient stress on expression of genes[1-6].Different stress-regulating pathwaysform complexregulatory networks through certain intermediate signal factors,such as hormones,active oxygen,calcium and some special proteins.14-3-3 proteins are an important class of regulatory proteins.They can directly regulate the expression of genes involved in some special resistances and signaling pathways.In addition,they can also participate in some metabolic pathways and change the orientation of some transcription factors as complex ligands[7].

The first 14-3-3 protein was found in rice callus under salt stress[8].In tobacco,14-3-3 protein is also found to be have similar functions[9].InArabidopsis,two 14-3-3 proteins are capable of regulating the resistance of plants to low temperature stress,and they are named as RCI1 and RCI2[10].Their expression levels are increased with the decrease in temperature,and are unrelated with plant growth and development.The expression of RCI1 and RCI2 is also not induced by ABA,NaCl or water stress[10-11].InArabidop sis,there are at least 13 genes encoding 14-3-3 proteins.In other plants,the expression of 14-3-3-homologous genes is induced by slat[12],drought[13]and other abiotic stresses.It indicates that 14-3-3 is a common regulator in plants’responses to various abiotic stresses.

In recent years,the resistanceregulating mechanism of 14-3-3 genes has been extensively studied.The action patterns of14-3-3 includes phosphorylating 14-3-3 by activating kinase[14]and interacting with other pro teins[15].14-3-3 proteins are a class of phosphoserine binding proteins.They take part in signal transduction,cell cycle response and stress response[16].Due to participating in a variety of cellular processes,14-3-3 proteins are also known as general regulatory factors (GRFs).The 14-3-3 gene inGlycine soja,GsGF14o,is involved in plant development and drought stress resistance.The over-expression ofGsGF14oresults in decreased drought resistance ofArabidopsisduring seed germination and seedling growth.However,the silencing ofAtGF14μ,a most homologous resistance withGs-GF14o,increases the drought resistance ofArabidopsis.Other studies have shown that the over-expression ofGsGF14owill decrease the size of stomasandnetphotosyntheticrate and block the formation and development of root hairs,decreasing absorption capacity of roots,eventually restricting growth of plants under drought stress.The expression levels of drought resistance-associated genes are decreased in plants under drought stress[17].In forage crops,there are rare reports on 14-3-3 proteins.In this study,the expression profiles of tall fescue under drought stress were studied by proteomic method.The results showed that the expression level of 14-3-3 gene family in tall fescue was significantly induced.In addition,the expression levels of 14-3-3 genes in tall fescue under low nitrogen,high temperature,drought and high salt stresses were studied using fluorescence quantitative PCR.This study will provide certain material basis and technical reserve for further researches on 14-3-3 proteins in forage and breeding ofstress-resistantgermplasms by genetic modification.

Materials and Methods

Materials

Plant materialQiancao 1 is new nationalforage cultivarbred by the Guizhou Institute of Prataculture in 2005.Its registration number is 299.

Main reagentsThe inorganic salts forpreparing Hoagland hydroponic solution were purchased from the Shanghai Medicines&Health Products Imp.&Exp.Co.,Ltd.The RNA extraction kit was purchased from the Axygen Scientific Inc.The RNA reverse transcription kit was purchased from the Thermo Scientific Company.The fluorescence quantitative PCR kit was purchased from the Promega Corporation.TheTaqenzyme,yeast extract,peptone and agarose were all purchased from the Shanghai Sangon Biological Engineering Technology&Services Co.,Ltd.The agarose DNA extraction kit was purchased from the Omega BioTek Inc.The X-gal,IPTG,ampicillin and kanamycinwere purchased from the Shanghai Sangon Biological Engineering Technology&Services Co.,Ltd.

Methods

Preparation of abiotic stress treatment of plant materialTotal 300newly-harvestedFestuca arundinaceaseeds were selected and soaked in water at 50℃overnight.Subsequently,the seeds were soaked in 75%ethanol for 30 s for surface disinfection.And then,they were rinsed three times with sterile water.The filter paper was laid in petri dishes,and then 4-5 ml of sterile distilled water was added into eachpetri dish.The disinfected seeds were placed evenly on the filter paper,which was maintained moist by adding 3-4 ml of sterile distilled water every day.The seeds were inoculated in a light incubator(L∶D=16∶8,22 ℃)[18]for 7 d.After the germination,the seeds were transferred to the Hoagland solution.After a 30-d culture,the nitrogen-free treatment(nitrogen stress treatment,and the NO3-in the solution was replaced by Cl-)and normal nitrogen treatment(control treatment,and the NO3-concentration in the Hoagland solution was as usual)were performed for the seeds.The low nitrogen stress lasted for 15 d.For the high salt treatment,theFestuca arundinaceaplants were soaked in NaCl-contained(400 mmol/L)Hoagland solution and normal Hoagland solution for 24 h respectively 7 d after their germination.In the high temperature treatment group,the germinatedFestuca arundinaceaplants were transplanted in soil matrix and cultivated for 30 d,and then,they weretreated by high temperature (42℃)for 24 h.In the drought treatment group,theFestuca arundinaceaplants were soaked in 30%PEG solution for 24 h 7 d after their germination.The nutrient solution was replaced once every three days.At the same time,in order to reduce the effect of microenvironment in the greenhouse,the planting pots were rotated regularly.The Festuca arundinacea leaves in both treatment and control groupsweresampled on hour 0,0.5,1.0,2.0,6.0,12.0 and 24.0,respectively.They were rapidly frozen by liquid nitrogen and preserved at-80℃for use.

Extraction of total RNA from Festuca arundinacea and RT-PCR

The RNA inFestuca arundinacealeaves was extracted using the TaKaRaRNAiso Reagent Kit,and reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit[19].The extracted RNA was treated by 1 μl of DNase.For each sample,5 μg of RNA was reverse transcribed into cDNA.

Bioinformatic analysis of14-3-3 geneThe cell component prediction,biological process prediction and gene function cluster analysis were carried out for the 13 14-3-3 genes inArabidopsisby Gene Ontology.The cluster analysis and sequence alignment of the four 14-3-3 genes inFestuca arundinaceawere performed using-Clustal W2.0.

Fluorescence quantitative PCR analysis of 14-3-3 gene in Festuca arundinacea under abiotic stressesThe expression levels of 14-3-3 gene in Festuca arundinacea under low nitrogen,drought,high temperature and high salt stresses were detected by fluorescence quantitative PCR.The synthesizedcDNA wasdiluted20 times and used as the template for fluorescence quantitative PCR.The reaction procedure was as follows:95℃53 min,95℃10 s,58℃10 s,72℃30 s,45 cycles,72℃10 min.The temperature was slowly increased from 65℃to 95℃,and the fluorescence signal was collected once every 0.5℃for the production ofmelting curves.Based on the cDNA sequence of 14-3-3 gene inFestuca arundinaceaobtained by transcriptome sequencing,the primers for PCR were designed using an online software(http://www.idtdna.com/Scitools/Applications/Primerquest),including14-3-3B-F:5’-TAGTGATGAGTGGCGGTTTAGTTGA-3’and 14-3-3B-R:5’-ATAAGGTCATGCTTGCTGAAATCAA-3’;14-3-3C-F:5’-TTGCCTACCCTGGATAAGATCTAAG-3’and 14-3-3C-R:5’-TAATAAACCCAGTCGTATCGCTTAG-3’;14-3-3A-F:5’-ATGAGGGTGGTGATGAGATCAAG-3’and 14-3-3A-R:5’-CGAACAATACAGGTAGCTGCGAAT-3’;14-3-3D-F:5’-CAAGATGAAGGGCGACTACCA-3’and 14-3-3D-R:5’-GCCAAATCTGCAAGAGCGATG-3’.The reference gene was the UBI(UBI-F:5’-CACCTCGATCACCCACCTCT-3’and UBI-R:5’-AGGGTCTCCGATAACCTCC A-3’).The final expression levels of 14-3-3 genes inFestuca arundinaceawere calculated using the 2-ΔΔctmethod[19].There were three biological replicates for each sample,and there were three technical replicates for eachbiological replicate.

Results and Analysis

Bioinformatic analysis of14-3-3 gene family in Arabidopsis

InArabidopsis,total 13 14-3-3 genes were discovered.The cluster analysis was conducted for the 13 14-3-3 genes inArabidopsis.The results showed that inArabidopsis,22.45%of 14-3-3 geneswere located in cytoplasms,and 12.25%,10.2%and 10.2%of 14-3-3 genes were located in cells,membranes and nucleus(Fig.1a).The biological process prediction showed that 26.67%and 22.45%of 14-3-3 genes take part in unknown biological processes.In the known processes,13.33%and 10.67%of 14-3-3 genes take part in biotic and abiotic stresses(Fig.1a).The cellular function prediction showed that the 14-3-3 genes all can bind with proteins (77.78%)and nucleic acids(13.89%)(Fig.1c).The14-3-3 gene family is predicted as protein ligandsforregulatorycomplexes.

Sequence analysis of 14-3-3 genes intallfescue

Based on the cDNA sequences of 14-3-3 genes obtained by transcriptome sequencing,primers were designed to amplify the full-length cDNA sequences of the four 14-3-3 genes in tall fescue.The four 14-3-3 genes were named as 14-3-3A,14-3-3B,14-3-3C and 14-3-3D.To study the homologies between the four genes and functional differentiation,the evolutionary relationships between14-3-3A,14-3-3B,14-3-3C and 14-3-3D proteins were investigated using Clustal W2.0.The results showed that the 14-3-3A and 14-3-3D were classified into one group,and 14-3-3B and 14-3-3C were classified into one group,indicating closer genetic relationships between 14-3-3A and 14-3-3D,14-3-3B and 14-3-3C(Fig.2).The sequence alignment also confirmed this conclusion (Fig.3).The homologies between the four genes ranged from 76.9%to 88.8%.The different regions are mainly located in the C terminals (Fig.3),indicating similar functions of the genes.The small differences in function may be mainly derived from the differences in sequences of Cterminals.

Expression characteristics of 14-3-3 genes in tall fescue under abiotic stresses

Previous studies have found that the expression of 14-3-3 genes can respond to a variety of different environmental factors.The transcriptional and proteomic studies also show that the expression of 14-3-3 genes in tall fescue can also respond to low nitrogen stress.To investigate whether 14-3-3 genes in tall fescue make response to different environmental stresses,the changes in expression levelsof 14-3-3 genes in tall fescue were studied under lownitrogen,hightemperature,drought and high saltstresses.

Under low nitrogen stress,the expression levels of 14-3-3A and 14-3-3 C were increased significantly 0.5 h after the starting of stress (Fig.4 a,Fig.4c);and the expression level of 14-3-3B was increased significantly 1 h after the starting of stress(Fig.4b).The expression of the three genes all rapidly responds to low nitrogen stress.In the late period (2-24 h),the expression levels of 14-3-3A,14-3-3B and 14-3-3C were all remained stable or decreased slightly.However,the variation trend of expression level of 14-3-3D was exactlydifferent.The expression of 14-3-3D was significantly inhibited in the very early period(0.5-1.0 h),but was significantly induced in the late period(12-24 h)(Fig.4d).

Under drought stress,the expression level of 14-3-3A was increased first and then decreased.It reached the peak 1 h after the starting of stress,and returned to the original level after 12 h;in the late period(12-24 h),the expression level of 14-3-3A was remained unchanged.The expression of 14-3-3B was induced rapidly.Its expression level was increased by four times in the stress interval of 0.5-1.0 h,and returned to the original level after two hours.The expression of 14-3-3C was induced instantaneously or slowly.Its expression level was increased rapidly 0.5-1.0 h after the starting of stress,and was increased slowly in the stress interval of 2-24 h.Similar to low nitrogenstress,the expression level of 14-3-3D was affected slightly by drought stress.The expression level of 14-3-3D differed insignificantly during the whole stressperiod.

Under condition of high temperature stress,the expression of 14-3-3A was induced significantly 1 h after the beginningof stress,and it returned to the original level after two hours.In the middle and late stress period(6-24 h),the expression of 14-3-3A was significantly induced again,and it was remained till the end of the stress.High temperature stress showed slight effect on expression level of 14-3-3B.There were no significant differences in expression level of 14-3-3B between the treatment and control groups during the stress interval of 0-20 h.However,its expression was significantly induced 24 h after the beginning of stress.Different from those of most 14-3-3 genes,the expression levels of 14-3-3C and 14-3-3D were inhibited in varying degrees by high temperature stress,which lasted for the whole stressperiod.

The expression level of 14-3-3A was increased significantly 1 h after the starting of high salt stress,and then rapidly returned to the original level.Different from that of 14-3-3A,the expressionlevelsof 14-3-3B,14-3-3Cand 14-3-3D were affected insignificantly by high salt stress.There were no significant differences in expression levels of 14-3-3B,14-3-3C and 14-3-3D between treatmentandcontrolgroups.

Conclusions and Discussion

14-3-3 genes are a class of relatively conserved genes in plants and animals.Previous cluster analysis has classified 14-3-3 genes into four groups,plant group,fungi group,alveolate group and animal group,and this is synchronous with entire biological evolution.It indicates that 14-3-3 genes are a class of very ancient resistance genes[20-21].In different organisms,the evolution of 14-3-3 gene is independent,thereby forming a variety of function-differentiated genes.In the model plantArabidopsis,total13 14-3-3 genes have been discovered,and in tall fescue,total four 14-3-3 genes have been successfully isolated.

In plant cells,14-3-3 proteins are involved in responses to a variety of stresses,such as high temperature,low temperature,drought and high salt.They improve stress resistance of plants mainly through increasing contents and activities of 14-3-3 proteins.The 14-3-3 proteins can enhance the activity of ATPase through binding with H+-ATPase complex.They can also enhance the resistance of plants by phosphorylating the C-terminal of proton pump located in cell membrane.The binding capacity of 14-3-3 proteins is induced and regulated by blue light.The 14-3-3 proteins are also closely related with opening and closure of stomas controlled by potassium ion.In addition to H+and K+,14-3-3 proteins are also closely related with the calci-um-dependent protein kinase(CPK)[22].Recent studies have found that in rice,14-3-3 proteins participate in regulation of plant height by interacting with brassinosteroid(BR)[23].However,in tobacco,14-3-3 proteins are found to be involved in the responses of plants to low temperature and high salt stresses and effects of ABA,ethylene and other plant hormoneson plants.

14-3-3 proteins can not only participate in the regulation of plants to abioticstresses,but also to be able to regulate plant resistance to diseases and pests.In barely,it is found that the expression of 14-3-3 gene is induced by non-host powdery mildew by subtractive hybridization,and its expression is increased first and then decreased.Similar results have been reported inArabidopsis thaliana,tomato,soybean and other crops.In this study,the expression of 14-3-3 genes in tall fescue can be induced or inhibited by many abiotic stresses,and the response speeds of 14-3-3 genes to abiotic stresses are different,indicating the functions of 14-3-3 genes have been differentiated in tall fescue,similar to other species.The 14-3-3 proteins participate in different stress regulations or different stages of the same stress regulation.Thisstudywillprovide theoretical basis for breeding of stress-resistant forage crops,especially tall fescue germplasms.

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