Advances in Cold Tolerance Genes and Their Application in Genetic Engineering of Plant for Cold Tolerance

Junzhi DUAN,Ying LI,Lei ZHOU,Yinghua PAN,Mingzhong ZHAO,Yinling REN*

1.Institute of Agricultural Economy and Information,Henan Academy of Agricultural Science,Zhengzhou 450002,China;2.Henan Agricultural University,Zhengzhou 450002,China;3.Institute of Food Crops,Hubei Academy of Agricultural Sciences,Wuhan 430064,China;4.Rice Research Institute,Guangxi Zhuang Autonomous Region Academy of Agricultural Sciences,Nanning 530005,China;5.Wheat Research Institute,Henan Academy of Agricultural Science,Zhengzhou 450002,China

Low temperature is one of the main environmental stress factors influencing plantgrowth and development and crop yield[1].Because tolerance to low temperature in plants is a quantitative trait controlled by many genes and its mechanism is complicated,progress in the development of improving cold tolerance in crops by traditional breeding methods has been time consuming,energy wasting and difficult.Therefore,with the development of molecular biology,molecular biological techniques had been applied to the research about plant adverse resistance continuously.Considerable attention has been paid to exploring excellent gene resource by modern molecular biological techniques and improving the cold tolerance in plants by way of genetic engineering.It is well known that under low-temperature stress conditions proteins functioning to protect plant directly are called functional proteins,and the genes coding such proteins are known as functional genes,which are the most downstream genes in the signaling pathway of low-temperature stress,such as genes involved in biosynthesis of osmotic adjustment substance including proline,glycine betaine and micromolecule sacccharides,genes coding antifreeze protein,genes coding key enzymes for fatty acid desaturation including GPAT and FAD,genes coding antioxidant enzymes including SOD,APX,GR and GST and genes coding LEA,COR and HSP.This study comprehensively and systematicallyreviewed these cold tolerance gene and progress of their application in genetic engineering of plant for cold tolerance,aiming increasing the resource for cold tolerance genes and laying the foundation for cold tolerance genetic improvement and breeding of plant.

Genes Involved in Biosyn-Thesis of Osmotic Substances

In low-temperature stress,plants avoid low-temperature injury by induction synthesis of osmotic substances maintaining osmotic pressure balance,among which proline,glycine betaine,trehalose and levan play an important role in tolerance to low temperature in plants.

Transforming a key enzyme for proline degradation-antisense gene of proline dehydrogenase,AtproDH,intoArabidopsis thaliana,could well control the yield of proline dehydrogenase,thus improving the intracellular proline content and enhancing the tolerance to low temperature and high salt in plants[1].In rice,the key geneOsP5-CS2 for proline synthesis lost its function through insertion of T-DNA,resulting in a mutant,which was sensitive to salt and cold compared with a wild type,and grew slowly after the treatment of high salt and low temperature,indicating thatOsP5CS2 was very important for tolerance to cold and salt in rice[2].OverexpressingVigna aconitifolia P5CSgene in larch resulted in the transgenic plants with improved tolerance to frost and salt[3].

Glycinebetaine biosynthetic pathway includes choline dehydrogenation/oxidation and glycine methylation.The former one exists in most plants,animals and microorganisms,and generates glycinebetaine via one or two steps of dehydrogenation/oxidation,involving the enzymes including choline monooxygenase(CMO),betaine aldehyde dehydrogenase (BADH)and choline oxidase (COD).The latter pathway,which was found inAphanothece halophytica,takes glycine as a substrate and produces GB via three steps of methylation involving two enzymes which are glycine sarcosine methyltransferase(GSMT)and sarcosine dimethylglycine methyltransferase (SDMT)[4].There were reports finding that:overexpression ofCodAgene inArabidopsis thalianacould improve betaine content in transgenic plants,thereby improving the freezing tolerance in plants[5-6];overexpression ofCodAgene in rice not only could improve the tolerance of transgenic rice to cold,but also could improve the tolerance to salt in transgenic rice[7];overexpression ofAtriplex hortensis BADHgene in wheat not only could improve integrity of membrane of transgenic wheat and reduce reactive oxygen content and degree of membrane lipid peroxidation,but also could improve the tolerance to cold in transgenic wheat[8];and overexpression ofAphanothecehalophyticaApGSMTandApDMTgenes in rice could improve the tolerance to cold and salt in transgenic rice[4].

Fusion of trehalose-6-phosphate synthase (TPS)and trehalose-6-phosphate phosphatase(TPP),which are key enzyme for trehalose synthesis inEscherichia coligenes,produces TPSP gene,the overexpression of which can improve trehalose content in transgenic rice,thereby enhancing tolerance to low temperature,drought and high salt in transgenic rice[9-10].Liet al.cloned in rice theOsTPS1 gene,which had TPS activity solely and was overexpressed in rice,discovering that trehalose and proline contents and the expression quantities of some stressrelated genes increased,resulting in improved tolerance to cold,salt and drought in plants[11].

In addition,introducingSacBgene coding levansucrase cloned in and isolated fromBacillus subtilisinto tomato improved levan contentin transgenic tomato and thus improved cold tolerance in plants[12];and transformingWFT2 gene coding levansucrase of wheat into rice improves levan content in transgenic rice plants as well as cold tolerance in plants[13].Galactinol synthase (GS)is a key enzyme in the synthesis of oligosaccharides of raffinose family,and transforming AmGS expressed in Ammopiptanthus mongolicusafter cold inducing treatment intoPhotinia serrulatatree improved the tolerance of transgenic Photinia serrulata plants to cold[14].

GenesCodingAntifreeze Proteins

Antifreeze proteins (AFPs)are a class of proteins having heat stagnation effect and ice crystal growth inhibitory effects,which reduce the freezing point of a water solution in nonlinear form but hardly influence melting point,resulting in a difference value between the freezing point and the melting point of the water solution,and thus help organisms to resist freezing conditions under low-temperature stress[15].

Antifreeze proteins were first found in fishes and insects.Wallis et al.transformed the fusion gene of a signal peptide of kidney bean phytolectin andPseudopleuronectes americanusAFP into potato,it was found that the transformants had an obviously-reduced leaf electrolyte leakage at-2℃compared with nontransgenic plants and thus were endowed with improved freezing tolerance,and furthermore,the AFP protein expression level in the transformants was positively associated with the freezing tolerance in the transformants[16].Huanget al.directed the gene coding Pseudopleuronectes americanus AFP into tomato,finding that the transgenic plants exhibited a growth status better than that of the controlunderfield low-temperature conditions,and a lethal temperature decreased by 2℃compared with the control[17].Similarly,by transforming the gene codingChoristoneura fumiferanalarva AFP intoVolvariella volvacea,it was found that the transgenic plants has good low-temperature tolerance which could be inherited stably[18];and by transforming the gene codingMicrodera punctipennisdzungarica AFP into tobacco,the transformants obtained obvious-improved cold tolerance[19].

Later,AFPs were found in plants.CarrotAFPgene was expressed after cold induction,and by overexpression of the gene inArabidopsis thaliana,the protein extract of the transgenic plants had obvious antifreeze activity,which was positively correlated withAFPgene transcription level[20];and overexpression of carrot AFP gene in tobacco improved cold tolerance in the transgenic tobacco[15,21].By transforming the gene coding winter rye AFP intoArabidopsis thaliana,the transgenicArabidopsisthalianaobtained enhanced membrane stability and improved cold tolerance under low-temperature stress conditions[22].Trans-formingAmmopiptanthus mongolicus AnAFPgene into tobacco provided the transgenic tobacco which had the relative electrolytic leakage with little change and showed wilting phenomenon rarely[23].These results indicates that AFP genes have potential application value in genetic development of important crops for cold tolerance.

Gene Coding Key Enzymes for Fatty Acid Desaturation

Biological membrane is the main partsuffering hilling damage,and the primary reaction happened with the phase change of lipoid molecules of biological membrane system[24].In 1973,Lyons put forward the hypothesis of“cold injury of membrane phase change”,in which he deemed that:when plants suffered low-temperature injury,it was biological membranes who suffered at first membrane lipid phase transformation from liquid crystalline phase into gel phase,the fatty acid chains on membrane lipids changed from unordered arrangement into ordered arrangement,on the membranes occurred channels and fractures resulting in increased membrane permeability,and a lot of soluble substances within the membranes leaked out of the membranes,damaging the ionic equilibrium between intracellular and extracellular environments;and membrane bound enzyme structure changed as well,and enzymatic reaction speed lost its equilibrium,resulting in physiological metabolism changes and dysfunction in plant cells,and finally injury in plant cells[25].There were researches that found,the lower the phase-transition temperature of plants,the better the cold tolerance;and the unsaturation degrees of lipoid and fatty acids in membrane lipids could obviously influence phase-transition temperature of membrane lipids,and higher unsaturation degree resulted in lower phase-transition temperature which then led to increased cold tolerance in plants[26-28].

Therefore,genetic engineering techniques can be applied to introducing genes coding enzymes for fatty acid desaturation into plants,to improve tolerance to cold in plants by reducing the saturation degree of fatty acids.The key enzymes for fatty acid desaturation mainly includes glycerol-3-phosphate acyltransferase(GPAT)gene,ω-3 fatty acid desaturase(FAD)gene,Stearoyl-acylcarrier protein(ACP)desaturase (SAD)gene and Δ6,Δ9,Δ12 desaturase genes.

Phosphatidylglycerol (PG)including more saturated fatty acids is the main factor deciding membrane phase transition,andGPATis the first acyl-esterifying enzyme in the biosynthesis of PG,and plays a vital role in deciding the unsaturation degree of membrane PG[29].By introducing the GPAT gene of cold-sensitive pumpkin into tobacco,the saturation degree of fatty acids in the membrane lipids of transgenic tobacco increased,while by introducing theGPATgene of coldtolerantArabidopsis thalianainto tobacco,the fatty acid composition in thylakoid PG of the transgenic plants tended to be unsaturated resulting in greatly-increased tolerance to cold in transgenic plants[26].Similarly,introducing theGPATgene ofArabidopsis thalianainto rice improved unsaturated fatty acid content and photosynthetic rate in leaves of transgenic rice,and the cold tolerance in the transgenic plants increased[30].Ariizumiet al.directedAGPATandSGPATgenes ofArabidopsis thalianaand spinach into rice,respectively,and found that the contents of cis unsaturated fatty acids of PG in the leaves of the two kinds of transgenic rice both increased obviously,and their photosynthetic rates both increased,so the transgenic rice grew at accelerated rates and had improved cold tolerance[31].

There have been many researches on ω-3 fatty acid desaturase(FAD)besidesGPAT.By constitutive overexpression of chloroplastFAD7 fromArabidopsis thalianain tobacco,the transgenic tobacco had increased triethenoid fatty acid content and correspondingly-reduced precursor substances thereof,exhibiting obvious cold tolerance,indicating thatthe plant ability of adapting and resisting low temperature could be improved through fatty acid desaturation[32-33];and furthermore,by expression of the same after induction by low-temperature stress,the transgenic tobacco also had increased triethenoid fatty acid content,and its survival rate was significantly improved,indicating great cold tolerance[34].Besides theFAD7 gene,Gibsonet al.isolated fromArabidopsis thalianaanother chloroplast gene (FAD8)coding enzyme for ω-3 fatty acid desaturation,which had nucleotide sequence homology reached 75% withFAD7 gene,and the two genes had complementary functions and co-catalyzed fatty acid desaturation in membrane lipid[35].It was found by the research onBnFAD8 gene ofBrassica napusthat this gene was expressed in a trace quantity under room temperature buthad a greatly-increased expression quantity in leaves under a low temperature condition,and it was thus speculated that theBnFAD8 gene was related to the low-temperature regulation inBrassica napus[36].In addition,the research on chloroplastCachFADgene in pepper found that,when leaves was injured, the transcription level ofCachFADgene was improved rapidly and then the content of linolenic acid(18:3)was improved as well[37];and transforming tobacco microsome gene NtFAD3 into sweet potato improved the content of linolenic acid (18:3)in transgenic sweet potato[38].Further research found that overexpression of tomato endoplasmic reticulumLeFAD3 gene in tomato not only improved the content of linolenic acid(18:3)in transgenic tomato,but also reduced the degree of cold injury in transgenic tomato[39].

Some progress also has been made in the researches on Δ6, Δ9 andΔ12 desaturases and stearoyl ACP desaturase.Four genes,desA,desB,desCanddesDfor desaturases isolated fromSynechocystissp.PCC 6803,coded proteins which specifically catalyzed the desaturation at Δ12,ω3,Δ9 andΔ6 positions of fatty acids,respectively,and had their expression depended on low temperature to varying degrees.Underlow-temperature stress conditions,expression quantity of desA increased,and the degree of unsaturation of membrane lipids increased,resulting in increased mobility of membrane lipid;the expression levels of genes for Δ6 and ω3 desaturases also increased at different increas-ing speeds;and the expression level of Δ9 desaturase hardly changed[40-42].By transforming Δ9 desaturase genedesCcloned in yeast or cyanobacteria into tobacco,the leaves of the transgenic tobacco had increased unsaturated fatty acid content,and the transgenic plants had obviously-increased cold tolerance[43].In addition,Maet al.directed gene SAD for spinach stearoyl ACP desaturase into tobacco,and found that the cold tolerance of the transgenic tobacco was enhanced[44];and the research on potato stearoyl ACP desaturase Δ9 gene found thatunderlow-temperature stress,the expression quantity of the gene in cold-tolerant variety was improved,while the expression quantity thereof in cold-susceptible variety did not changed,so Δ9 gene was related to the tolerance to cold in potato[45].

Genes for Antioxidant Enzymes

In case of abiotic stress,a lot of reactive oxide species(ROS)would be generated in cells of plants,resulting in oxidative stress,membrane lipid peroxidation and membrane protein aggregation,and membrane permeability increases,such that normal membrane structure is damaged.Plants form a complicated antioxidant protective system for clearing away or reducing ROS injury in a long evolutionary process,including a non-enzymatic protective system and an enzymatic protective system.The non-enzymatic protective system includes glutathione(GSH),carotenoid (CAR),ascorbic acid,etc.;and the enzymatic protective system includes superoxide dismutase(SOD),catalase(CAT),peroxidase (POD),acorbateperoxidase(APX),etc.The overall antioxidant protective system involves antioxidantdetoxification enzymes including SOD,CAT,POD,APX,monodehydroascorbate reductase(MDHAR),dehydroascorbate reductase (DHAR),glutathione reductase(GR),glutathion peroxidase(GPX),glutathione s-transferase(GST)and aldose/aldehyde reductase(ALR).

SOD is the first key antioxidant enzyme for clearing away ROS,and it can clear away superoxide radicals and derivatives thereof generated by cells subjected to stress.Introducing Mn-SOD cDNA from tobacco into alfalfa constructed two plasmid vectors,pMitSOD and pChlSOD,the pMitSOD containing a transit peptide sequence carried Mn-SOD protease and entered mitochondria,the synthetic protein coded by pChlSOD was targeted to chloroplast,and it was found that the transgenic plants had enhanced resistance to herbicide diphenyl ether and enhanced tolerance to low-temperature stress and the plants with Mn-SOD transgene targeted to different organelles including mitochondria and chloroplast had different resistances[46].Duanet al.found by overexpressing a tomato thylakoidal ascorbate peroxidase geneLetAPXinto tomato,under cold stress conditions,the transgenic tomato had improved APX activity,chlorophyll content,net photosynthetic rate and maximum photochemical efficiency and reduced hydrogen peroxide content,electrolyte leakage and MDA content and the overexpression ofLetAPXalleviated photoinhibition in the transgenicplantsand improved cold tolerance in the transgenic plants[47].By overexpression of turnipMDHARgene in Arabidopsis thaliana,the expression quantities of genes for APX,DHAR,GR,SOD,GPX and so on in the transgenic arabidopsis thaliana increased,the contents of ASA,GSH and chlorophyll contentwereimproved,andMDA content was reduced,resulting in enhanced cold tolerance in the transgenic plants[48].Reduction in expression quantity of tomatoMDHARgene by RNAi technique reduced MDHAR activity,and then the cold tolerance in transgenic tomato also was enhanced[49].Shuet al.isolated tomatoLeGRgene and obtained antisense transgenic tomato,which compared with non-transgenic tomato had more accumulated hydrogen peroxide,serious electrolyte leakage and decreased net photosynthetic rate and maximum photochemical efficiency,resulting in inhibited growth and development and reduced APX activity,GSH content and AsA content,and they found the transgenic plants were sensitive to cold,soLeGRgene could enhance cold tolerance in plants[50].The detoxication of plasma membrane peroxidation products with higher toxicity such as HNE (4-hydroxynon-2-enal)is completed by bonding with GSH,which is completed through the catalysis by GST and ALR.There were reports showing that overexpression of ALR gene could enhance cold tolerance and cadmium tolerance in transgenic tobacco[51];and transforming riceOsGSTL2 gene intoArabidopsis thalianaenhanced the resistance of the transgenicArabidopsis thalianato heavy metal,cold,salt and osmotic stress[52].

Genes Coding LEA,COR and Other Functional Proteins

Genes coding abundantLEA protein,COR protein and dehydrin for late embryogenesis are closely related to cold tolerance in plants,and almost all of the 3 kinds of proteins are hydrophilic and can alleviate cell injury caused by salt,drought,freezing and dehydration.LEA protein is a kind of protein accumulated in late embryogenesis ofplants,has high hydrophilicity and thermal stability,and is closely related to plant stress resistance.Overexpression of tomato LEA protein geneLe25 in yeast notably improved tolerance of transgenic yeast cells to cold and salt[53];and overexpression of barley LEA protein geneHVA1 in mulberry improved the cold and salt tolerance and drought resistance in the transgenic mulberry[54].

COR protein genes are cold inducible mostly,for example,COR15a,COR6.6,COR78,COR47homologous toLEAand so on are generated in cold domestication process ofArabidopsis thaliana,and some COR proteins have the typical structure of the proteins ofLEAfamily and thus are endowed with high hydrophilicity capable of reducing the injury to plants caused by dehydration.Constitutive expression ofCOR15ainArabidopsis thalianain a large amount obtained the transgenic plants,which had enhanced cold tolerance in chloroplast and protoplast as well as enhanced stability of plasma membrane compared with the wild type,resulting in alleviated injury caused by low tempera-ture[55].TheBnCOR25 gene isolated fromBrassica napuswas expressed in hypocotyls,cotyledon,stems and flowers in lager expression quantities which were affected by low temperature and osmotic stress;and by transformingthe geneintoArabidopsis thaliana,the rooting speed of Arabidopsis thaliana was rapider than that of wild plants at a low temperature of 4℃,indicating that overexpression ofBnCOR25 gene could improve the cold tolerance in transgenic yeast andArabidopsis thaliana[56].Leaf mustardCbCOR15bgene which was expressed in maturation zones of roots,stems and leaves was adopted to transform tobacco,and from the points of electrolyte leakage,relative water content,glucose content and phenotype,the transgenic tobacco suffered lighter injury under cold stress and had enhanced cold tolerance[57].Dehydrin has lysine-enriched structural domain as well as high hydrophilicity,played a role of molecular chaperone in response to stress,and can alleviate the injury to plants caused by dehydration stress.Potato dehydrin geneDhn24 which was expressed in all of roots,stems,leaves and cotyledons was transformed into cucumber,and it was found that the transgenic cucumber had alleviated cold injury and enhanced cold tolerance,but the degree of tolerance to cold was not related to the expression level ofDHN24 protein[58].Hoiet al.researched the dehydrin geneDprCisolated fromAspergillusfumigatusand found its deletion mutant had decreased cold tolerance,the gene could enhance cold tolerance in plants,and the DprC protein-mediated cold tolerance response depended on SakA MAP kinase[59].

In addition,there were some functional protein genes which participated in cold stress response,such as hestshockproteins (HSPs)and aquaporins.A lot of HSP shave molecular chaperone activity,and can maintain the function structures of proteins,thereby preventing misfolded proteinsand dysfunctionalproteins from aggregating.By transforming paprikeCaHSP26 gene coding chloroplast small heat shock protein into tobacco,it was found that the overexpression of this gene improved the photochemical efficiency and cold tolerance in the transgenic tobacco under cold stress[60].Aquaporins are a kind of proteins located on cell membranes,form“passages”on cell membranes and can control water entering or exiting cells.Cotton GhTIP1;1 coding tonoplast aquaporin was overexpressed in yeast,and the yeast cells had improved survival rate under cold stress,indicating that the gene was related to cell cold tolerance[61].In addition,Qiaoet al.transformed animal cell death inhibitor geneBcl-xLinto potato and found thatBcl-xLcould inhibit cell death by maintain the dynamic equilibrium of organelles,thereby enhancing the viability of plants under cold stress conditions[62].Thorlbyet al.foundArabidopsis thalianaβ-glucosidase geneSFR2mutant had increased electrolyte leakage under lowtemperature stress conditions and was sensitive to cold,gene knockout could achieve the same result,indicating thatSFR2 gene could enhance cold tolerance in plants,and it was speculated thatitmightparticipate in polysaccharide conversion in cell walls and protection of cell membranes from low-temperature injury[63].Komoriet al.showed in their research that male sterile restoring geneRf-1 could enhance the fertility of hybrid rice under low-temperature stress[64].Xuet al.found thatArabidopsis thaliana AZI1 gene was expressed with the induction of azelaic acid and low temperature,and by overexpressing it inArabidopsis thaliana,the low-temperature injury was alleviated in cells of the transgenic plants;and if the gene was knocked out,the transgenic plant suffered aggravated low-temperature injury,and if the gene was introduced intoSaccharomyces cerevisiae,the survival rate ofthe transgenic Saccharomyces cerevisiae increased under low temperature stress,indicatingAZI1 could enhance cold tolerance in plants[65].Zhanget al.found rice vacuole H+-translocating inorganic pyrophosphatase geneOVP1 was expressed after low temperature induction;and overexpression of this gene in rice improved the cold tolerance in the transgenic rice,and the transgenic rice had increased cellmembrane integrity,decreased MDA contentand increased proline content[66].Zhouet al.transformed tomato carotenoid ε-hydrozylase geneLeLUT1 into tobacco,and found that the transgenic tobacco had reduced reactive oxygen content,MDA content and electrolyte leakage,improved photochemicalefficiency and net photosynthetic rate and enhanced cold tolerance[67].

Prospects

With global climate change,especially in the context of gradually warming global climate,extreme climates occurs more frequently,and the frequency of low-temperature disaster caused by uneven cold and heat,rain,snow and freezing weather increases.Therefore,improving cold tolerance in crops become more and more important in the aspects of maintaining high and stable yield and sustainable development in agriculture and ensuring national food security.Currently,Some progress has been made in the aspect of functional genes for cold tolerance,such as genes for osmotic adjustment substance,antifreeze protein,key enzymes for fatty acid desaturation and antioxidant enzymes by genetic engineering.However,tolerance to low temperature is controlled by many genes and inherited as a quantitative trait,the transgenic plants obtained by genetic engineering are single transgenic plants mostly,less of them are double transgenic plants,and cold tolerance in transgenic plants can be improved in a limited range.Therefore,considerable attention will be paid to systematic study on the transformation of more related genes to obtain ideal cold-tolerant plants in future.At present,the technique of transforming multiple genes is not mature yet,and further research is necessary.

In addition,the identification of cold tolerance in transgenic plants is conducted under controllable indoor conditions mostly,while natural field cold disaster is different with indoor simulated cold disaster and the transgenic plants showing cold tolerance under indoor conditions may not have cold tolerance under field conditions.Therefore,on the basis of indoor identification of cold tolerance in transgenic plants, further field identification should be performed to determine the cold tolerance of transgenic plants.It should be pointed out that genes for cold tolerance driven by constitutive strong promoter will cause dwarf and dysontogenesis of plants while improving cold tolerance in transgenic plants,which are likely to affect crop yield.Genes for cold tolerance driven by stress-inducible promoter can be used in future studies,and cold tolerance in transgenic plants should be improved withoutaffecting normal growth and development of plants.

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