Gene Regulation During Cold Stress Acclimation

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ERF转录因子研究进展

ERF转录因子研究进展高浩;竺锡武【摘要】ERF(Ethylene-responsive factor)转录因子是AP2/ERF大家族中的一个大的亚家族,仅含1个AP2/ERF结构域,每个成员都含有1个由大约60个氨基酸组成的非常保守的DNA结合域.有研究表明,每种植物有100种以上ERF转录因子,其功能各不相同,分别具有调节植物生长发育、抗生物胁迫和非生物胁迫的作用等.本文就ERF转录因子的研究现状及发展趋势进行分析,以期为ERF转录因子的应用提供参考.【期刊名称】《现代农业科技》【年(卷),期】2017(000)008【总页数】3页(P130-131,134)【关键词】ERF转录因子;功能;作用机理【作者】高浩;竺锡武【作者单位】湖南人文科技学院,湖南娄底 417000;湖南人文科技学院,湖南娄底417000【正文语种】中文【中图分类】Q943随着环境条件的恶化，植物在生长发育过程中受到的非生物因素和生物因素影响会更多，如高温、低温、干旱、盐碱、病虫害等。

在不断适应环境和进化过程中，植物形成了复杂有效的逆境胁迫应答体系，可以调节植物使其能够适应新的生长环境。

其中，在转录水平的调控过程中转录因子发挥了非常重要的作用[1]。

转录因子又称反式作用因子，是一群能与真核基因启动子区域中的顺式作用元件发生特异性结合，从而保证目的基因以特定的强度、在特定的时间与空间表达的蛋白质分子[2]。

AP2/ERF家族转录因子对植物非常重要，可以调控植物整个生命周期的生长发育和逆境[3-7]。

根据AP2结构域的数目和结构特点，AP2/ERF家族转录因子可分为4个亚族（ERF、DREB、AP2、RAV）和单独成员（Soloist）[4-5，8]。

ERF（Ethylene-responsive factor）转录因子是 AP2/ERF大家族中的一个大的亚家族，仅含1个AP2/ERF结构域，在结构域序列的第14位和第19位分别是丙氨酸和天冬氨酸。

植物抗寒性的生理生态学机制研究进展_徐燕

第43卷第4期2007年4月林业科学SCIE NTIA SILVAE SINICAE Vol .43,No .4Apr .,2007植物抗寒性的生理生态学机制研究进展＊徐　燕　薛　立　屈　明(华南农业大学林学院　广州510642)摘　要:　综述植物在冷驯化过程中发生的一系列生理生化变化。

环境对植物抗寒性的影响主要与光诱导、温湿度以及气候的变化有关。

植物表面形成冰层会引起植物的无氧呼吸,导致植物受害;光抑制诱导活性氧的产生,从而导致植物光合系统的退化,抗寒能力下降,而短日照诱导植物休眠,有利于植物抗寒。

光敏色素则被认为是启动冷驯化的光受体;植物通过冷驯化增加碳水化合物的积累及病原体相关蛋白的合成,以增强对低温病原体的抵抗能力;气候的变化使植物遭受了更大的冷伤害风险。

微管最初遇冷时部分的解体可以有效诱导植物抗寒性;抗氧化酶活性增强,植物体内糖、脯氨酸、多胺等内含物含量上升。

植物休眠状态中的生理变化(种子的休眠、芽的休眠)与AB A 敏感性的差异有关。

对植物抗寒性分子机制的研究表明:CO R 基因的表达对于植物抗寒性和冷驯化是十分关键的;与气候梯度有关的基因梯度的分布说明寒冷地区的树种更为抗寒;多表型性状的数量性状分析,为重要的农艺性状标记辅助选择(MAS )提供基础。

对植物抗寒过程中的信号转导进行研究发现,Ca 2+是低温下参与调节冷驯化应答机制中信号转导途径的重要的第二信使。

未来植物抗寒领域的研究热点为信号转导和基因调节,低温抗性的遗传学和遗传应用及代谢组学,气候变化对于植物抗寒的影响等方面。

关键词:　植物;环境;抗寒;生理;分子机制中图分类号:S718.43;Q945.78 文献标识码:A 文章编号:1001-7488(2007)04-0088-07收稿日期:2006-01-20。

基金项目:广东省林业局项目(4400-F02084,4400-F05004)。

＊薛立为通讯作者。

Physiological and Ecological Mechanisms of Plant Adaptation to Low TemperatureXu Yan Xue Li Qu Ming(C oll eg e o f For es tr y ,Sou th Chi na Agr icul tur al Uni vers ity Gu ang zh o u 510642)Abstract :　Chilling injury is one of the ecological factors c ausing environmental stress in plants .Exploring the physiological and ecological mechanisms of c old tolerance in plants can under stand ho w plants gro w at low temperatur e ,which has important meaning in theory and practice .At present ,study on cold toler ance in plants focuses on physiology ,genes ,and relationship between plants and environment and so on .This article revie ws the physiological and ecological response of plants to lo w temper atur e during c old acclimation .Over wintering plants encased in ice can be exposed to anaerobic conditions and suffer phytotoxicity .Photoinhibition induced the increase of r eactive oxygen species (ROS ),causing the degradation of photosystems ,which is unfa vorable for cold hardiness of plants .Shor t da ys induc e dor manc y in plants ,resulting in a increase in cold hardiness of plants .Phytochr ome has been c onsidered to be the photoreceptor r esponsible for tr iggering the initiation of the first sta ge of c old acclimation .The acc umulation of carbohydrates and pathogenesis -related proteins enhances the resistance of plants to low -temper atur e pathogens .Scientists pr edict that plants will suffer greater risk of low -te mperature da mage with the c hanges in climate .An initial partial disassembly of microtubles is sufficient to trigger efficient cold acclimation .The adaptation of plants to c old also associates with the incr eased levels of antioxidants enzymes ,sugar ,proline ,polymines and so on .Changes in dormanc y status are mor e likely related to changes in ABA sensitivity than to var iations in ABA levels .The expr ession of COR (cold r esponsive )genes is cr itical in plants for both c hilling tolerance and c old acclimation .Genotypes fr om colder envir onments have greater cold hardiness in situ than those from milder envir onments .The QTL analysis of multiple phenotypic traits pr ovides the basis for marker assisted selection (MAS )of important a gronomic characters .Calcium is an important second messenger in a low temper atur e signal transduction pathway involved in regulation of c old -acclimation response .Signal tr ansduction ,gene re gulation ,genetics ,metabolomics ,and climate change affecting the plant survival are impor tant aspects in the future study of c old tolerance in plants .Key words :　plants ;environment ;cold tolerance ;physiology ;molec ular mechanism低温寒害是农林业生产中一种严重的自然灾害,据统计,世界每年因此造成的损失达2000亿美元(卢存福,2004)。

不饱和脂肪酸在逆境胁迫中的作用_年洪娟

【收稿日期】2012-02-07【基金项目】国家自然基金（31160020）和云南省自然科学基金（2009ZC014X ）资助【作者简介】年洪娟，女，副教授，硕士生导师，从事微生物抗逆分子生物学研究，Email ：nianhongjuan@yahoo．com．cn文章编号：1005-376X （2012）08-0760-03【综述】不饱和脂肪酸在逆境胁迫中的作用年洪娟，陈丽梅（昆明理工大学生物工程技术研究中心，昆明650500）【摘要】生物膜是将细胞与环境分开的第一道屏障，是环境胁迫造成损伤的主要位点。

脂肪酸是生物膜的主要组成成分，不饱和脂肪酸在决定生物膜的生理特性中具有重要作用，增加脂肪酸的不饱和程度能增加膜脂的流动性。

近年来，很多研究发现，生物通过脂肪酸脱饱和维持膜的流动性来适应外界环境变化。

本文主要从不饱和脂肪酸在环境温度胁迫、盐胁迫、氧化胁迫、酸碱胁迫、干旱胁迫、乙醇胁迫及铝胁迫中的作用研究进展进行了综述。

【关键词】不饱和脂肪酸；胁迫【中图分类号】Q945；X173【文献标识码】AThe role of unsaturated fatty acids in various environmental stressesNIAN Hong-juan ，CHEN Li-mei（Biotechnology Research Center ，Kunming University of Science and Technology ，Kunming 650500，China ）【Abstract 】Biological membranes are the first barrier that separates cells from the environment and are a primary target for damage during environmental stress．Fatty acids are the major components of cell membranes．Unsaturated fatty acids play an important role in the biophysical characteristics of cell membranes．An increased degree of unsaturation increases fluidization of the membrane lipids．Recently ，many researches proved that organisms usually maintain the fluidization of membrane by de-saturation of the fatty acids to adapt to the environmental changes．This paper reviewed the recent progresses on the roles of un-saturated fatty acids in various stresses ，such as environmental temperature stress ，salt stress ，oxidative stress ，acid and alka-line pH tolerance ，drought tolerance ，ethanol tolerance and aluminum stress．【Key words 】Unsaturated fatty acids ；Stress生物将自身与环境分开的第一道屏障是生物膜，生物膜也是环境胁迫过程中发生损害的主要位点。

苏州医工所研究生导师简介

苏州医工所研究生导师简介导师介绍张春，博士，于一九八五年获南京师范大学生物学学士学位，一九九二年获中国科学院遗传研究所遗传学硕士学位，二00一年获美国佛罗里达大学分子和细胞生物学博士学位。

二00一年八月到二00二年九月在佛罗里达大学医学系从事博士后研究。

二00二年九月到二00三年十月在佛罗里达大学药学系继续从事博士后研究。

自二00三年十月，被佛罗里达大学分子遗传及微生物系聘为助理科学家。

自从二00二年九月，开始从事基因治疗的研究。

在佛罗里达大学药学系的工作期间，成功地用非病毒载体（多聚乙氨，PEI）将基因转入哺乳动物脑细胞里去。

自二00三年十月，在美国科学院院士Berns 博士的实验室工作。

在工作期间，研发了一种用腺相关病毒（AAV）作为载体的基因定点整合系统。

这是一种能将外源基因安全、有效、永久、位点特异性地转入到人类细胞１９号染色体上特定位点(AAVS1)并允许基因长期表达的方法。

这项顶尖技术将会带来基因治疗安全用于人类的突破。

二00九年七月，中国医学科学院医学生物学研究所，研究员，学科带头人，基因疫苗实验室主任。

二0一0年八月至今，中国科学院苏州生物医学工程技术研究所，研究员，分子诊断和治疗实验室主任。

二0一二年七月，实验室获批为苏州市分子诊断和治疗技术重点实验室，并任实验室主任。

实验室现在有实验员两名，博士后两名，博士研究生一名，硕士研究生两名，联合培养硕士研究生七名。

实验室现在承担和开展的项目有：1，用rAAV位点特异性整合载体建立一株治疗帕金森氏症的分泌神经生长因子（GDNF）的成人脑神经星状前体细胞的细胞株30970880，国家自然科学基金，面上项目，2010年1月1日-2012年12月31日，项目主持；2，哺乳动物细胞合成生产抗凝血因子FVIII，Y157011705，苏州生物医学工程技术研究所，创新项目，2011年10月1日-2013年10月31日，项目主持；3，基于腺相关病毒（AAV）倒置末端重复序列（ITR）的基因表达微载体，江苏省基础研究计划（自然科学基金）--面上研究项目，2012-07-01 至 2015-07-01，项目主持；4，苏州市分子诊断和治疗技术重点实验室，科技基础设施建设计划，2011年7月1日-2014年6月30日，项目主持；5，便携式细菌内毒素快速检测仪及检测试剂，ZXY2012011，医疗器械与新医药专项2012年7月1日- 2015年6月30日，项目主持。

植物在低温胁迫下的过氧化氢代谢及信号转导

( PHT /1999 /081 ) ; 公益性行业科学专项
© 1994-2010 China Academic Journal Electronic Publishing House. All rights reserved.
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园艺学报
© 1994-2010 China Academic Journal Electronic Publishing House. All rights re迫下的过氧化氢代谢及信号转导
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量显著下降。由于 APX和 CAT对 H2 O2 的亲和力不同 , 有人认为 APX可能对 H2 O2 信号调控起作用 , 而 CAT才是胁迫条件下针对 H2 O2 的过量产生起调节作用的物质 (M ittler, 2002) 。郝晶等 ( 2007) 研究表明 , 低温下 ( 6 ℃) 大豆幼苗 H2 O2 含量呈上升趋势 , 胁迫初期低浓度 H2 O2 诱导了 CAT、 POD 等保护酶活性的提高。张立军等 (2007) 在低温下采用不同浓度的外源 H2 O2 处理大豆种子 , 结果也表明适宜浓度的 H2O2 可以诱导 CAT、 POD 等保护酶活性增加。
Key words: hydrogen peroxide; low temperature stress; signal transduction; cold resistant
过氧化氢 (Hydrogen peroxide, H2 O2 ) 是生物细胞代谢过程中产生的一种活性氧。正常情况下 , 植物体内 ROS清除系统能够使其含量保持在伤害水平之下 ; 但是逆境胁迫下 , 活性氧的产生量会超过系统清除能力 , 由此造成氧化损伤。在过去的很长一段时间 , 人们将 H2 O2视为一种有害的细胞代谢产物。然而 , 越来越多的研究证明 , 植物可以利用 H2 O2并将其放大作为信号分子 , 进而调控生长发育过程和对各种生物和非生物刺激的应答。各种生物和非生物胁迫均能在一定水平上诱导细胞中 H2 O2的产生 , 进而调控一系列胁迫应答的信号转导。

（细胞生物学专业优秀论文）组蛋白乙酰化修饰调控果蝇热休克基因表达和寿..

摘要衰老是一个普遍的生物学现象，衰老控制着生物寿命的长短，主要受遗传因子和环境因素所影响。

了解衰老的分子机制，对于延缓衰老、保持生命活力具有重要的意义。

热休克蛋白（ＨＳＰ）作为高度保守的“分子伴侣”，在细胞内广泛地参与许多复杂的功能活动，可以抵制衰老过程中一些有害蛋白的发生。

其基因的表达调控是一种特殊的真核基因表达模式，包括基础水平和诱导水平的表达。

由组蛋白乙酰转移酶（ＨＡＴ）和组蛋白去乙酰化酶（ＨＤＡＣ）催化的乙酰化反应在真核基因的表达调控中起着重要作用，这两种酶通过对核心组蛋白进行可逆修饰来调节核心组蛋白的乙酰化水平，从而调控转录的起始与延伸。

组蛋白去乙酰化酶抑制剂（ＨＤＩ）可以通过抑制ＨＤＡＣ活性提高组蛋白乙酰化水平，是研究乙酰化修饰在真核基因表达调控中的作用的有用工具。

本论文一方面采用ＨＤＩｔｒｉｃｈｏｓｔａｔｉｎＡ＜ＴＳＡ）和丁酸钠（ＢｕＡ）喂食果蝇，改变果蝇体内组蛋白乙酰化水平，系统地研究组蛋白乙酰化修饰、ＨＳＰ的表达以及寿命调控三者之间的关系。

结果发现ｈｓｐ基因在长寿果蝇中具有较高的基础表达、较快的热激诱导反应速度以及较强的高温抵抗性。

同时，不同的ｈｓｐ基因在果蝇衰老过程中的作用不尽相同，ｈｓｐ２２的作用最为重要，ｈｓｐ７０次之，而ｈｓｐ２６的表达几乎与寿命无关。

使用ＨＤＩＴＳＡ和ＢｕＡ喂食果蝇可以延长其寿命，但不同的ＨＤＩ的作用机制不尽相同，同一种ＨＤＩ对不同寿命品系的果蝇的延长程度也不尽相同。

ＴＳＡ的处理有一种时间依赖性，更长时间的ＴＳＡ处理对寿命是有利的；而ＢｕＡ的处理却与此不同，过长时间的处理反而加速衰老。

同样的去乙酰化酶抑制剂，同一剂量处理，在不同果蝇品系种的作用不同，它们对短寿果蝇寿命的延长程度更为明显。

另外，ＨＤＩ处理还促进果蝇衰老过程中ｈｓｐ基因的基础表达和诱导表达，但是随着衰老的进行，这种促进作用逐渐减弱。

同样在不同寿命的果蝇品系中，其提高ｈｓｐ基因表达的程度也不一样。

冷热应激对肉牛生理指标及基因表达影响的研究进展

冷热应激对肉牛生理指标及基因表达影响的研究进展应激反应是指动物在受到外界频率较大,持续时间较长(或短但变化剧烈)的刺激时,机体内环境稳定性、生理指标等发生改变,进而影响到机体健康程度的一种反应,产生的主要原因包括极端温度、运输和转场等方面[1],而由外界温度变化产生的冷热应激,是诸多应激反应中较为常见的一种,可从多个方面对机体健康产生不利影响。

在肉牛产业中,饲养环节既是基础部分,又是最重要的环节,而在饲养过程中产生的冷热应激,可对肉牛的能量代谢产生影响并降低群体健康状况。

已有研究发现,在热应激状态下肉牛群体的采食量、生长效率较非应激状态普遍降低[2-3],而严重的冷应激也会对干物质摄入量(DMI)和耗水量产生负面影响[4]。

总之,冷热应激可从生产效能、存活率、繁殖率等多个方面造成经济损失,进而影响肉牛产业的发展。

所以,如何降低冷热应激对机体产生的影响,改善肉牛冷热应激情况下的健康状况,是肉牛产业发展亟需解决的问题。

在动物体内,生理生化和免疫指标可表征机体的生理功能状况与健康程度。

在应激状态下,动物个体的内环境稳定性会发生变化,进而造成血液生理生化和免疫指标较非应激状态下出现显著差异[5]。

本文从冷热应激的评判标准和产生地域入手,基于冷热应激的产生机制,对冷热应激下不同地区、不同品种的肉牛血液内分泌、免疫、抗氧化等生理生化指标变化情况进行概述,挖掘对冷热应激起到调控作用的基因和调控因子,讨论并展望了缓解肉牛冷热应激的可行性方法,从而为促进我国肉牛产业的高质量发展提供理论参考。

1 冷热应激的评判标准及调控机理1.1 肉牛冷热应激的评判标准外界温度变化是否对机体产生冷热应激的辨别标准包括温湿度指数(temperature humidity index,THI)和综合气候指数(comprehensive climatic index,CCI)等[6-7]。

THI可基于温度和湿度评价肉牛机体应激状况,其计算公式为THI=(Td + Tw) + 40.6(Td为干球温度;Tw为湿球温度),当THI<72时,肉牛群体为无应激状态;当THI>72时,可分为轻度(72～79)、中度(79～88)和严重热应激(大于88)3个程度。

基因相关词汇专业英语翻译

基因相关词汇专业英语翻译Aactivation domain 活化结构域adapters 连接物adenine 腺嘌呤adenosine 腺ADP (adenosine diphosphate) 腺二磷酸affinity column 亲和柱AFLP (amplified fragment length polymorphisms) 增值性断片长度多态现象agrobacterium 农杆菌属alanine 丙氨酸allele 等位基因amber mutation 琥珀型突变AMP (adenosine monophosphate) 腺一磷酸ampicillin 氨青霉素anchor primer 锚状引物annealing 退火annealing temperature 退火温度anticodon 反密码子AP-PCR (arbitrarily primed PCR) 任意引物聚合链反应arbitrary primer 任意引物ATP (adenosine triphosphate) 腺三磷酸autosome 常染色体Bbaculovirus 杆状病毒base pair 基对base sequence 基顺序beta-galactosidase β-半乳糖beta-glucuronidase β-葡糖醛酸糖bioluminescence 生物发光bioremediation 生物降解biotechnology 生物技术blotting 印迹法blue-white selection 蓝白斑筛选blunt end 平(整末)端Ccatalyst 催化剂cDNA library 反向转录DNA库centromere 着丝体centrosome 中心体chemiluminescence 化学发光chiasma 交叉chromomere 染色粒chromoplast 有色体chromosomal aberration 染色体畸变chromosomal duplication 染色体复制chromosomal fibre 染色体牵丝chromosome 染色体chromosome complement 染色体组chromosome map 染色体图chromosome mutation 染色体突变clone 克隆cloning 无性繁殖系化codon 密码子codon degeneracy 密码简并codon usage 密码子选择cohesive end 黏性末端complementary DNA (cDNA) 反向转录DNA complementary gene 互补基因consensus sequence 共有序列construct 组成cosmids 黏性质粒crossing over 互换cyclic AMP (cAMP) 环腺酸cytosine 胞嘧啶Ddark band 暗带deamination 脱氨基作用decarboxylation 脱羧基作用degenerate code 简并密码degenerate PCR 退化性聚合链反应dehydrogenase 脱氢denaturation 变性deoxyribonucleoside diphospahte 脱氧核糖核一磷酸deoxyribonucleoside monophospahte 脱氧核糖核二磷酸deoxyribonucleoside triphospahte 脱氧核糖核三磷酸deoxyribose 去(脱)氧核糖dicarboxylic acid 二羧酸digoxigenin 洋地黄毒diploid 二倍体DNA (deoxyribonucleic acid) 去(脱)氧核糖核酸DNA binding domain DNA结合性结构域DNA fingerprinting DNA指纹图谱DNA helicase DNA解螺旋DNA kinase DNA激DNA ligase DNA连接DNA polymer DNA聚合物DNA polymerase DNA聚合double helix 双螺旋double-strand 双链Eelectroporation 电穿孔endonuclease 内切核酸enhancer 增强子enterokinase 肠激episome 游离基因ethidium bromide 溴乙锭eukaryotic 真核生物的euploid 整倍体exonuclease 外切核酸expressed-sequence tags 表达的序列标记片段extron 外含子FF factor F因子FAD (flavine adenine dinucleotide) 黄素腺嘌呤二(双)核酸feedback control 反馈控制feedback inhibition 反馈抑制feedback mechanism 反馈机制first filial (F1) generation 第一子代FISH (fluoresence in situ hybridization) 荧光原位杂交forward mutation 正向突变F-pilus F纤毛functional complementation 功能性互补作用fusion protein 融合蛋白Ggel electrophoresis 凝胶电泳gene 基因gene cloning 基因克隆gene conversion 基因转变gene duplication 基因复制gene flow 基因流动gene gun 基因枪gene interaction 基因相互作用gene locus 基因位点gene mutation 基因突变gene regulation 基因调节gene segregation 基因分离gene therapy 基因治疗geneome 基因组/ 染色体组genetic map 基因图genetic modified foods (GM foods) 基因食物genetics 遗传学genetypic ratio 基因型比/ 基因型比值genome 基因组/ 染色体组genomic library 基因组文库genotype 基因型giant chromosome 巨染色体globulin 球蛋白glucose-6-phosphate dehydrogenase 6-磷酸葡萄糖脱氢GP (glycerate phosphate) 磷酸甘油酸脂GTP (guanine triphosphate) 鸟三磷酸guanine 鸟嘌呤Hhaploid 单倍体haploid generation 单倍世代heredity 遗传heterochromatin 异染色质Hfr strain 高频重组菌株holoenzyme 全homologous 同源的housekeeping gene 家务基因hybridization 杂交Iimmunoglobulin 免疫球蛋白in vitro 在体外/ 在试管内in vivio 在体内independent assortment 独立分配induced mutation 诱发性突变induction 诱导initiation codon 起始密码子inosine 次黄insert 插入片段insertional inactivation 插入失活interference 干扰intergenic 基因间的interphase 间期intragenic 基因内的intron 内含子inversion 倒位isocaudarner 同尾酸isoschizomer 同切点JKkanamycin 卡那毒素klenow fragment 克列诺夫片段Llac operon 乳糖操纵子ligase 连接ligation 连接作用light band 明带linker 连接体liposome 脂质体locus 位点Mmap distance 图距离map unit 图距单位mature transcript 成熟转录物metaphase 中期methylase 甲基化methylation 甲基化作用microarray 微列microinjection 微注射missense mutation 错差突变molecular genetics 分子遗传学monoploid 单倍体monosome 单染色体messenger RNA (mRNA) 信使RNAmultiple alleles 复(多)等位基因mutagen 诱变剂mutagenesis 诱变mutant 突变体mutant gene 突变基因mutant strain 突变株mutation 突变mutation rate 突变率muton 突变子NNAD (nicotinamide adenine dinucleotide) 烟醯胺腺嘌呤二核酸NADP (nicotinamide adenine dinucleotide phosphate) 烟醯胺腺嘌呤二核酸磷酸nicking activity 切割活性nonsense codon 无意义密码子nonsense mutation 无意义突变Northern blot Northern印迹法nuclear DNA 核DNAnuclear gene 核基因nuclease 核酸nucleic acid 核酸nucleoside 核nucleoside triphosphate 核三磷酸nucleotidase 核酸nucleotide 核酸nucleotide sequence 核酸序列Ooligonucleotide 寡核酸one gene one polypeptide hypothesis 一个基因一种学说operon 操纵子oxidative decarboxylation 氧化脱羧作用oxidative phosphorylation 氧化磷酸化作用PPCR (polymerase chain reaction) 聚合链反应peptidepeptide bond 键phagemids 噬菌粒phosphorylation 磷酸化作用physical map 物理图谱plasmid 质粒point mutation 点突变poly(A) tail poly(A)尾polymerase 聚合polyploid 多倍体positional cloning 位置性无性繁殖系化primary transcript 初级转录物primer 引物probe 探针prokaryotic 原核的promoter 启动子protease 蛋白purine 嘌呤pyrimidine 嘧啶QRrandom segregation 随机分离RAPD (rapid amplified polymorphic DNA) 快速扩增多态DNAreading frame 阅读码框recessive gene 隐性基因recombinant 重组体recombinant DNA technology 重组DNA技术recombination 重组regulator (gene) 调控基因replica 复制物/ 印模replica plating 复制平皿(板)培养法replication 复制replication origin 复制起点reporter gene 报道基因repression 阻遏repressor 阻遏物repressor gene 阻遏基因resistance strain 抗药性菌株restriction 限制作用restriction enzyme 限制性内切restriction mapping 限制性内切图谱retrovirus 反转录病毒reverse transcription 反转录作用RFLP (restricted fragment length polymorphisms) 限制性断片长度多态现象ribonucleotide 核糖核酸ribose 核糖ribosomal RNA (rRNA) 核糖体RNAribosome 核糖体RNA (ribonucleic acid) 核糖核酸RNA polymerase I RNA聚合IRNA polymerase II RNA聚合IIRNA polymerase III RNA聚合IIIR-plasmid R质粒/ 抗药性质粒Ssecond filial (F2) generation 第二子代self-ligation 自我连接作用shuttle vectors 穿梭载体sigma factor σ因子single nucleotide polymorphism 单核酸多态性single-stranded DNA 单链DNAsister chromatid 姊妹染色单体sister chromosome 姊妹染色体site-directed mutagenesis 定点诱变somatic cell 体细胞Southern blot Southern印迹法splice 拼接star activity 星号活性stationary phase 静止生长期sticky end 黏性末端stop codon 终止密码子structural gene 结构基因supernatant 上层清液supressor 抑制基因Ttelophase 末期template 模板terminator 终止子tetracycline 四环素thymine 胸腺嘧啶tissue culture 组织培养transcription 转录作用transfer RNA (tRNA) 转移RNA transformation 转化作用transgene 转基因translation 翻译/ 平移transmembrane 跨膜triplet 三联体triplet code 三联体密码triploid 三倍体UVvector 载体WWestern blot Western印迹法Aalternative splicing -- Eukaryotic genes are composed of exons and introns, the latter being removed by RNA splicing before transcribed mRNA leaves the nucleus. Commonly, a single gene can encode several different mRNA transcripts, caused by cell- or tissue-specific combination of different exons. This is known as alternative splicing.Annealing -- The time- and temperature-dependent process by which two complementary single-stranded polynucleotides associate to form a double helix (see also hybridization)Antisense strand -- the DNA strand of a gene which, during transcription, is used as a template by RNA polymerase to synthesize a complementary RNA strand.反股-- 意指一股DN**段为基因之所在，因此可用来当做模版使得RNA反转录脢在转录RNA时，可以合成和此DN**段完全结合的RN**段。

低温诱导的植物基因表达与调控

低温诱导的植物基因表达与调控杭州外国语学校（310023）周筱娟摘要低温是影响植物生长、发育和地理分布的重要因素。

近年来，大量研究发现低温诱导许多基因的表达，根据基因表达的蛋白产物，可分为编码功能蛋白基因和调节蛋白基因两大类。

本文对这两类低温反应基因的表达与调控及在低温胁迫中作用的最新研究进展进行介绍。

关键词：低温反应基因，低温驯化，基因表达低温是影响植物生长、发育及其地理分布的重要环境限制因素之一。

大多数热带和亚热带植物由于缺乏对低温的适应能力，当环境温度低于10℃时就会受到伤害，严重影响植物的正常生长、发育甚至造成死亡。

分布于温带地区的植物，在温暖季节对冰冻的抗性相当弱。

但是，随着季节的变化，气温的逐渐降低，植物对冰冻的抗性也逐渐增强。

在非冻的低温环境生长一段时间后，植物增强了抗冻能力，从而能耐受随即发生的冰冻温度，这个适应过程称为低温驯化（cold acclimation）。

根据植物的种类，达到最大抗冻性的低温驯化时间从数天至数周不等，不同种类植物可以耐受-10℃至-60℃以下的温度（Webb, Uemura & Steponkus 1994）。

因此，低温驯化是植物提高抗冻性的有效途径。

低温驯化是一个十分复杂的过程。

近二十年来，世界各地的科研工作者围绕在低温驯化过程中植物发生的生理生化和分子水平的各种变化进行了大量的研究。

最新的研究表明至少有300个低温反应基因参与了低温驯化进程（Fowler & Thomashow, 2002）。

针对如此复杂的适应过程，低温驯化研究的一个基本目标是分离和鉴定对抗冻性提高起着关键作用的低温反应基因。

随着突变分析和分子遗传学方法的大量应用，以拟南芥作为模式植物，已克隆了许多低温反应基因及低温调节的转录因子基因，明确了这些基因的抗冻功能及其涉及的多种低温调控的信号传导途径。

根据低温反应基因的蛋白产物可分为两大类：一类是直接保护细胞免受胁迫伤害的功能蛋白；另一类是传递信号和调控基因表达的调节蛋白。

Epigenetic regulation of stress responses in plants-2009

Available online at Epigenetic regulation of stress responses in plants Viswanathan Chinnusamy1and Jian-Kang Zhu2Gene expression driven by developmental and stress cues often depends on nucleosome histone post-translationalmodiﬁcations and sometimes on DNA methylation.A number of studies have shown that these DNA and histone modiﬁcations play a key role in gene expression and plant development under stress.Most of these stress-induced modiﬁcations are reset to the basal level once the stress is relieved,while some of the modiﬁcations may be stable,that is,may be carried forward as ‘stress memory’and may be inherited across mitotic or even meiotic cell divisions.Epigenetic stress memory may help plants more effectively cope with subsequent stresses. Comparative studies on stress-responsive epigenomes and transcriptomes will enhance our understanding of stress adaptation of plants.Addresses1Water Technology Centre,Indian Agricultural Research Institute,New Delhi110012,India2Department of Botany and Plant Sciences,Institute for Integrative Genome Biology,University of California,Riverside,CA92521,USACorresponding author:Zhu,Jian-Kang(jian-kang.zhu@)Current Opinion in Plant Biology2009,12:133–139This review comes from a themed issue onGenome studies and molecular geneticsEdited by Masahiro Yano and Roberto TuberosaAvailable online27th January20091369-5266/$–see front matter#2008Elsevier Ltd.All rights reserved.DOI10.1016/j.pbi.2008.12.006IntroductionInformation content of the genome(DNA sequence)and its expression in response to stress are crucial for the adaptability of a genotype.Expression of the genome is inﬂuenced by chromatin structure,which is governed by processes often associated with epigenetic regulation, namely histone variants,histone post-translational modi-ﬁcations,and DNA methylation.Developmental and environmental signals can induce epigenetic modiﬁ-cations in the genome,and thus,the single genome in a plant cell gives rise to multiple epigenomes in response to developmental and environmental cues[1].Under-standing stress-induced epigenetic processes in stress tolerance of plants requires answers to the following questions:How much of the stress-induced gene expres-sion changes are associated with alterations in DNA methylation and histone modiﬁcation marks?Are stress-induced DNA and histone modiﬁcations during acclimation or during theﬁrst experience of stress mem-orized and inherited mitotically and meiotically?What are the adaptive values of epigenetic stress memory?This review brieﬂy describes epigenetic processes,and then focuses on recent data on the epigenetic regulation of stress responses and its heritability in plants. Epigenetic regulation of stress responses Retention of stress memory for short durations is well known in plants,as evident from acclimation responses [2,3].The stress memory can be retained for only short durations if the memory depends on the half-life of stress-induced proteins,RNAs,and metabolites,while the memory can last longer if it involves reprogramming in phenology and morphology of plants.Epigenetic processes,that is,stable or heritable DNA methylation and histone modiﬁcations,can also be a choice of retaining stress memory for longer times.Methods to decipher epigenetic changes are brieﬂy described in Box1.Histone modiﬁcationsN-terminal regions of nucleosome core complex histones undergo various post-translational modiﬁcations.In addition,each histone has variants encoded by different genes.The combinations of histone variants and post-translational modiﬁcations can be considered a‘histone code’,which plays a key role in chromatin structure and thus determines the transcriptional state and expression level of genes.Some histone modiﬁcations,namely acety-lation,and certain phosphorylation and ubiquitination [4,5],enhance transcription,while biotinylation and sumoylation repress gene expression[6,7].Trimethyla-tion of H3K4activates transcription,while dimethylation of H3K9and H3K27represses transcription[5].Because several of the histone modiﬁcations are associated with changes in gene transcription in general,it is not surpris-ing that stress-induced gene regulation is associated with histone modiﬁcations in all cases that have been inves-tigated.Changes in histone variants,histone modiﬁ-cations as well as DNA methylation are often referred to as epigenetic regulation.However,such changes may or may not be truly epigenetic in nature because common epigenetics deﬁnition requires mitotic or meiotic herit-ability.Drought induced the linker histone variant H1-S in tomato.H1-S appears to be involved in the negative regulation of stomatal conductance,because stomatal con-ductance and transpiration rates were higher in antisense transgenic H1-S tomato plants than in wild type(WT) plants[8].In rice seedlings,submergence induced histone H3K4 trimethylation and H3acetylation in alcohol dehydrogen-ase1(ADH1)and pyruvate decarboxylase1(PDC1) genes.These histone modiﬁcations were correlated with enhanced expression of ADH1and PDC1under stress. The modiﬁcations,however,were dynamic and were restored to the basal level after stress was relieved by reaeration[9 ].Environmental and endogenous signals can repress the target genes through reduction in histone acetylation levels.The REDUCED POTASSIUM DEPEN-DENCY3(RPD3)family histone deacetylases(HDACs), namely HDA6and HDA19,mediate histone deacetyla-tion in response to biotic and abiotic stresses in Arabi-dopsis.HDA6is induced by jasmonic acid(JA)and ethylene[10].HDA6is involved in transcriptional gene silencing(TGS)[11]and RNA-directed DNA methyl-ation(RdDM)in Arabidopsis[12].Wounding,infection by Alternaria brassicicola,and plant hormones(JA and ethyl-ene)induced the expression of the HDA19/HD1/ AtRPD3A gene.Overexpression of HDA19in transgenic plants reduced histone acetylation levels and increased the expression levels of ETHYLENE RESPONSE FAC-TOR-1(ERF1)and PATHOGENESIS-RELATED(PR)genes.In contrast,RPD3A-RNAi plants exhibited higher histone acetylation,which was accompanied by down-regulation of ERF1and PR genes in Arabidopsis[10]. Enhanced HDA6and HDA19expression caused by stress and hormonal signals thus might affect chromatin modi-ﬁcations at several loci.ABA downregulated the expression of AtHD2C(a mem-ber of plant-speciﬁc HD2family of HDACs).Transgenic Arabidopsis plants overexpressing AtHD2C exhibited enhanced expression of ABA-responsive genes and greater salt and drought tolerance than the WT plants [13 ].In rice,expression of different members of the HDAC families is also differentially regulated by abiotic factors such as cold,osmotic and salt stress,and hormones such as ABA,JA,and salicylic acid[14].Besides the HDACs,the WD-40repeat protein TBL1 (T ransducin B eta-L ike protein-1)is associated with histone deacetylation in humans.The Arabidopsis hos15 (high expression of osmotic stress responsive genes15)mutant was hypersensitive to freezing stress,and was hypersen-sitive,in terms of germination,to ABA or NaCl.HOS15 encodes a protein similar to TBL1,which interacts with histone H4.HOS15is probably involved in H4deacety-lation because acetylated H4was higher in hos15mutants than in WT plants,and thus regulates stress tolerance through chromatin remodeling in Arabidopsis[15 ]. Drought-induced expression of stress-responsive genes is associated with an increase in H3K4trimethylation and H3K9acetylation in Arabidopsis[16 ].In Drosophila,H3 Ser-10phosphorylation activates transcription during heat shock responses[17].In Arabidopsis also,high sal-inity,cold stress,and ABA triggered rapid and transient upregulation of histone H3Ser-10phosphorylation,H3 phosphoacetylation,and H4acetylation followed by stress-type-speciﬁc gene expression[18 ].Histone acetyltransferases(HATs)interact with tran-scription factors and are involved in activating stress-responsive genes.GCN5is the catalytic subunit of the Spt-Ada-Gcn5acetyltransferase(SAGA)and transcrip-tional adaptor(ADA).Like ADA2and GCN5in the response of yeast to extreme temperature stress,in Ara-bidopsis as well,GCN5and ADA regulate cold tolerance by interacting with C-repeat B inding F actor-1(CBF1). CBF1activates transcription of its downstream cold-responsive genes probably through the recruitment of ADA/SAGA-like complexes that may mediate chromatin remodeling in target genes[19].DNA methylationDNA cytosine methylation,both asymmetric(m CpHpH)-methylation and symmetric(m CpG and m CpHpG)-meth-ylation,is associated with repressive chromatin in gene promoters and with repression of gene transcription.De134Genome studies and molecular geneticsBox1Deciphering epigenetic changesHistone modifications:Chromatin immunoprecipitation(ChiP)—histones bound to the DNA in vivo are covalently crosslinked to DNA in situ by vacuum infiltration of plant tissue with formaldehyde.Then chromatin is isolated as part of cell extract,fragmented,and protein–DNA complexes are immunoprecipitated with antibodies specific against modified histone,for example,acetylated or dimethylated H3K9. DNA is isolated from the immunoprecipitate and analyzed by PCR [4,9 ,15 ,16 ,18 ,51].ChiP-Seq—this method combines ChiP with next-generation sequencing technology such as Solexa sequencing to analyze gen-ome-wide-speciﬁc histone modiﬁcations[52].DNA methylation:Methylation-sensitive restriction endonucleases—the classical method of cytosine methylation analysis is the restriction analysis of template DNA with methylation-sensitive restriction enzymes.Re-stricted DNA is then ligated to restriction site specific adaptor and analyzed by PCR or restricted genomic DNA is analyzed by Southern blotting[22,27 ,49 ,50 ].Bisulﬁte method—sodium bisulﬁte converts cytosines,but not50-methylcytosines,into uracil,under denaturing conditions.PCR ampliﬁcation of bisulﬁte-treated DNA results in conversion of uracil to thymine.Bisulﬁte-treated DNA is analyzed by PCR or DNA sequen-cing[4,23 ,33 ,49 ,50 ]Methylated-DNA immunoprecipitation(MeDIP)—genomic DNA is fragmented and precipitated with5-methylcytosine-speciﬁc anti-body.The precipitated DNA is then analyzed by PCR or whole-genome tiling microarrays[53,54].Shotgun bisulﬁte-sequencing—this combines bisulﬁte treatment of genomic DNA with next generation sequencing technology such as Solexa sequencing.The converted sequences are mapped to the reference genome sequence to identify methyl-cytosines[21,55].novo methyltransferases DRM1(DOMAINS REARRANGED METHYLASE1)and DRM2catalyze new cytosine methylation,while the maintenance of symmetric CG and CHG methylation is mediated by the DNMT1-like enzyme MET1and the plant-speciﬁc enzyme Chromomethylase3(CMT3),respectively[20]. Recent studies suggested that MET1and CMT3may also catalyze de novo methylation,while DRM1and DRM2are also important for the maintenance of sym-metric methylation[1,21].Stresses can induce changes in gene expression through hypomethylation or hypermethylation of DNA.In maize roots,cold stress-induced expression of ZmMI1was cor-related with a reduction in methylation in the DNA of the nucleosome core.Even after seven days of recovery,cold-induced hypomethylation was not restored to the basal level[22].In tobacco,aluminum,paraquat,salt,and cold stresses induced-DNA demethylation in the coding sequence of the NtGPDL(a glycerophosphodiesterase-like protein)gene correlated with NtGDPL gene expres-sion[23 ].Osmotic stresses induced transient DNA hypermethyla-tion in two heterochromatic loci in tobacco cell-suspen-sion culture[24].DNA hypermethylation was also induced by drought stress in pea[25].In the facultative halophyte Mesembryanthemum crystallinum L.,drought and salt stresses-induced a switch in photosynthesis mode from C3to CAM.This metabolic change was associated with stress-induced-speciﬁc CpHpG-hypermethylation of satellite DNA[26].Transposons constitute a signiﬁcant portion of plant genomes and are maintained in a repressed state by DNA methylation.Environmental factors may activate transposons through DNA demethylation.In Antirrhinum majus,cold stress induced hypomethylation,and transpo-sition of the Tam-3transposon[27 ].Stress-induced histone modiﬁcations can also inﬂuence DNA methylation.Knockout mutants and RNAi lines of stress-inducible HDA6of Arabidopsis and HDA101of maize showed an increase in histone acetylation accom-panied by changes in histone methylation pattern and derepression of silenced genes[28,29].Speciﬁc histone modiﬁcation-dependent pathways appear to mediate methylation of about two-thirds of the methylated loci in the Arabidopsis genome[1].Thus,dynamic histone modiﬁcation marks could be converted into DNA meth-ylation marks,which are often more stable.RNA-directed DNA methylationGenetic analysis using Arabidopsis mutants impaired in genes for siRNA biogenesis or action revealed the invol-vement of small interfering RNAs(siRNAs)in RdDM [20,30].Integration of the Arabidopsisﬂoral epigenome with theﬂoral transcriptome and small RNA proﬁles revealed a direct correlation between the ability of geno-mic sequences to produce small RNAs and DNA meth-ylation[21].In fact,siRNAs are involved in the methylation of at least one-third of methylated loci [21].Studies on the repressor of silencing1(ros1)mutant of Arabidopsis revealed that the DNA glycosylase ROS1 actively demethylates DNA by a base excision repair mechanism and can counteract RdDM[31,32].ROS3, a RNA recognition motif-containing protein,binds to small RNAs and may direct sequence-speciﬁc demethyl-ation by ROS1and related DNA demethylases[33 ]. Gene silencing processes can be sensitive to temperature. Temperature and other abiotic stresses can also regulate speciﬁc small RNAs.Low temperature promoted virus-induced gene silencing,while high temperature delayed it[34].Endogenous siRNAs that are regulated by abiotic stress have been identiﬁed in Arabidopsis[35].In Arabi-dopsis,24-nt SRO5-P5CDH nat-siRNA downregulates the expression of P5CDH mRNAs through mRNA clea-vage,leading to decreased proline degradation,and enhanced proline accumulation and salt stress tolerance [36].This and other stress-regulated siRNAs conceivably could also lead to changes in histone modiﬁcations and DNA methylation.Microarray data showed that abiotic stresses and ABA inﬂuence the expression of many of the genes implicated in RdDM pathways in Arabidopsis(our unpublished data).Further studies are clearly needed to unravel the roles of RdDM pathway under stress. Plant development under stress Reprogramming of cell differentiation in response to environmental stress leads to phenological and develop-mental plasticity,which are important mechanisms of stress resistance.Phenotypic plasticity helps adjust the durations of various phenological phases in plants,and thus allows plants to avoid exposure of critical growth phases,and especially reproductive development,to stress.Further,adjustment of growth and development is critical for effective use of resources under stress. Germination and vegetative growthOsmotic stress reduces the uniformity of seed germina-tion and seedling establishment.Several HDACs are induced by ABA in Arabidopsis[13 ]and rice[14].Arabi-dopsis HDA19/HD1interacts with a global corepressor of transcription,AtSIN3,which in turn interacts with AtERF7(APETALA2/EREBP-type transcription fac-tor).Suppression of AtERF7and AtSIN3in plants caused hypersensitivity to ABA during germination and seedling growth[37].Arabidopsis HDA6/HDA19double repression lines showed growth arrest after germination and for-mation of embryo-like structures on true leaves[38]. These results suggest that ABA accumulation leads to change in expression or activity of HDACs,which in turn regulate growth under stress.Epigenetics of stress tolerance Chinnusamy and Zhu135Transgenic Arabidopsis overexpressing a SNF2/ BRAHMA-type chromatin remodeling gene AtCHR12 exhibited growth arrest of primary buds and growth reduction of the primary stem.These responses were more pronounced under drought and heat stress than under nonstress conditions.Conversely,the growth arrest response under stress was less in the AtCHR12-knockout mutant than in the WT plants[39 ].Reproductive developmentFlowering and seed development are crucial for plant reproduction.Hence,plants have evolved mechanisms toﬂower when environmental conditions are appropriate. In Arabidopsis,low temperatures during vernalization induce epigenetic mechanisms which repress the FLOW-ERING LOCUS C(FLC,a MADS-box protein)gene,and the repressed FLC chromatin is maintained till transition toﬂowering.The mechanisms of mitotic inheritance of the repressed epigenetic state of FLC chromatin and resetting during reproduction are not fully understood [40].Because the low temperatures that induce vernali-zation also induce cold acclimation,some of the gene expression programs could be under common epigenetic control.Mutations in some of the genes involved in stress-related epigenetic processes cause changes inﬂowering time. The hos15,a freezing sensitive mutant of Arabidopsis, was lateﬂowering owing to downregulation ofﬂower-ing-regulatory genes SOC and FT[15 ].Plant hormone and stress-regulated HDA6and HDA19may act as a link between stress and developmental cues that controlﬂow-ering and plant development.Reduction in HDA19 expression in antisense transgenic plants/T-DNA mutants resulted in developmental abnormalities in-cluding delayedﬂowering[41,42].HDA6-RNAi lines and axe1-5/hda6mutants showed hyperacetylation of histone H3globally,downregulation of JA-responsive genes,upregulation of FLC,and delayedﬂowering[43 ]. In Arabidopsis,FCA and FPA proteins form an autonom-ousﬂowering pathway by downregulatingﬂowering repressor FLC.Both FCA and FPA are RNA-binding proteins that can regulate DNA methylation[44].ABA and drought stress induced the expression of chromatin remodeling gene PsSNF5(Pisum sativum SNF5). PsSNF5interacts with Arabidopsis SWI3-like proteins (SWI3A and SWI3B),which in turn interact with FCA [45,46].ABA-induced SNF5and FCA may regulate ﬂowering time and stress responses through chromatin remodeling.Because stresses reduce crop yield and quality,and ABA regulates seed development partly through epigenetic processes[47],effects of stress on ABA accumulation or epigenetic processes therefore may affect seed/fruit development under stress.SenescenceAbiotic stresses induce premature leaf senescence,which leads to reduced photosynthesis and thus less biomass accumulation.JA–and ethylene-responsive-HDACs, HDA6and HAD19,appear to modulate leaf senescence. Arabidopsis HDA6-RNAi lines and axe1-5(hda6)mutants exhibited downregulation of JA-responsive genes and senescence-associated genes,and delayed senescence as indicated by higher chlorophyll content and PSII activity as compared to WT plants[43 ].In contrast, HDA19antisense transgenic plants/T-DNA mutants showed early senescence[41].Stress memoryUV-C radiation orﬂagellin(an elicitor of plant defense) induced a high frequency of somatic homologous recom-bination,and the hyper-recombination state was trans-mitted as a dominant trait to untreated progenies of stress-treated parents[48 ].Similarly,tobacco mosaic virus(TMV)infection resulted in a high frequency of somatic and meiotic recombination rates in tobacco.The progeny of TMV-infected plants exhibited hypomethyla-tion in several leucine-rich repeat(LRR)-containing loci and a higher frequency of recombination in hypomethyl-ated LRR-containing TMV(N-gene)resistant gene [49 ].The adaptive value of stress-induced epigenetic plasticity was studied in hypomethylation progenies of5-aza-deox-ycytidine(inhibitor of DNA cytosine methylation)-trea-ted rice seeds.In one of the progenies,methylation was completely erased in Xa21G,a Xa21-like protein gene. The erasure of promoter methylation and inheritance of this epigenetic state resulted in constitutive expression of Xa21G in the progeny line and enhanced resistance to the pathogen Xanthomonas oryzae pv.oryzae,race PR2[50 ]. ConclusionsStress-induced changes in histone variants,histone N-tail modiﬁcations,and DNA methylation have been shown to regulate stress-responsive gene expression and plant de-velopment under stress.Transient chromatin modiﬁ-cations mediate acclimation response.Heritable, epigenetic modiﬁcations may provide within-generation and transgenerational stress memory(Figure1).It is unclear how much of the stress-induced histone and DNA modiﬁcation changes that have been observed to date may be epigenetic in nature because little is known about their mitotic or meiotic heritability.Abiotic stress-induced epigenetic changes might have an adaptive advantage.However,stress memory could have a nega-tive impact on crop yield by preventing the plant from growing to its full potential.Thus,stress memory has implications for the use of seeds from stressed crop to raise ensuing crops by the farmers,breeding for stress environments and in situ conservation of plant species. 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植物低温信号的感知、转导与转录调控

中国农业科技导报,2009,11(3):5-11Journal of Agricultural Science and Technol ogy　收稿日期:2009202224;修回日期:2009203219　基金项目:国家973计划项目(2006CB100102);国家杰出青年基金(30525034)资助。

　作者简介:张融雪,硕士研究生,主要从事植物抗逆分子生物学研究。

Tel:010*********;E 2mail:zrx1230@ 。

通讯作者:黄荣峰,研究员,博士生导师,主要从事植物抗逆分子生物学研究。

Tel:010*********;E 2mail:rfhuang@caas .net .cn植物低温信号的感知、转导与转录调控张融雪1,2,　张治礼2,3,　张执金4,　黄荣峰4(1.海南大学农学院,海南儋州571737;2.中国热带农业科学院热带生物技术研究所,海口571101;3.海南省农业科学院,海口571000;4.中国农业科学院生物技术研究所,北京100081)摘　要:低温是植物生长的主要环境胁迫因子之一。

植物对低温的应激是一个复杂的过程,包括低温信号的感知、信号转导和转录调控等阶段。

低温可以通过质膜流动性的改变被质膜感知,也可以通过质膜上的钙离子通透性通道、组氨酸激酶、受体激酶和磷酸酯酶感知。

低温信号转导包括钙信号途径和其他信号途径,其中钙信号途径是低温应答过程中重要的信号途径。

在此途径中,因低温增加的胞质钙离子能被C DPK 、磷酸酶和MAPK 识别并传导;其他信号途径主要与ABA 有关。

低温信号最终将启动C BF 和非CBF 介导的转录调控,提高植物的低温抗性。

关键词:植物;低温;信号转导;转录调控中图分类号:Q756 文献标识码:A 文章编号:100820864(2009)0320005207S i gna l Percepti on,Tran sducti on and Tran scr i pti ona lRegul a ti on dur i n g Cold Stress i n Pl an tZHANG Rong 2xue1,2,ZHANG Zhi 2li 2,3,Z HANG Zhi 2jin 4,HUANG Rong 2feng4(1.College of Agriculture,Hainan University,Hainan Danzhou 571737; 2.I nstitute of Tr op ical B i oscience and B i otechnol ogy,Chinese Academy of Tr op ical Agricultural Sciences,Haikou 571101; 3.Hainan Acade my of Agricultural Sciences,Haikou 571000; 4.B i otechnol ogy Research I nstitute,Chinese Acade my of Agricultural Sciences,Beijing 100081,China )Abstract:Cold is one of the key envir on mental stress ors which affect p lant gr owth and devel opment .The res ponse p r ocess of p lant t o cold is a comp lex p r ocedure .It includes several step s,such as l ow te mperature signal percep ti on,signal transducti on,transcri p ti on regulati on .Cold can be perceived by p las ma membrane either due t o changes inme mbrane fluidity or with the hel p of sens ors like Ca 2+per meable channels,histidine kinases,recep t or kinases and phos pholi pases .Cold signal transducti on includes calciu m signal path way and other signal path ways,of whichcalciu m signal path way is an i m portant path way of cold res ponse p r ocess in p lant .I n this path way,cyt os olic Ca 2+induced by cold can be recognized and transduced by CDPKs,phoshatase and MAPKs .O ther signal path ways are mainly related t o ABA.Cold signal will at last s witch on C BF and non 2C BF independent transcri p ti onal cascade,thus t o i m p r ove p lant resistance t o cold .Key words:p lant;cold;signal transduti on;transcri p ti on regluati on 低温是主要的环境胁迫因子之一,能引起植物细胞膜脂相变、细胞水分亏缺、体内酶的活性降低和光合速率下降,严重时能形成胞外冰晶,刺伤细胞膜导致细胞破裂,从而影响植物的生长,制约植物的地域分布和生长季节,并且影响农作物的产量和品质。

生物技术概论论文-酵母基因工程菌的构建过程及其在食品领域中的应用

酵母基因工程菌的构建过程及其在食品领域中的应用随着科技的发展，食品生物技术在食品工业发展中的地位和作用越来越大，已经渗透到食品工业的方方面面，特别是基因工程技术等技术在21世纪的食品工业中充当重要的角色。

而工程菌就是用基因工程的方法，使外源基因得到高效表达的菌类细胞株系，是采用现代生物工程技术加工出来的新型微生物，具有多功能、高效和适应性强等特点。

主要应用于治理海洋石油泄漏，生产基因工程药物，酵母基因工程中等方面。

而酵母基因工程中，酵母基因工程菌就是菌类细胞株系用的是酵母菌，能够发挥着一定的功能，可以提高发酵的效率。

酵母基因工程的优点：1.是真核生物，大多具有价高的安全性。

2.繁殖速度快，能大规模生产，具有降低基因工程产品成本的潜力。

3.将原核生物中已知的分子和基因操作技术与真核生物中复杂的转运后修饰能力相结合，能方便外缘基因的操作。

4.采用高表达启动子，可高效表达目的基因，而且可诱导调控。

5.提供了翻译后加工和分泌的环境，使得产物和天然蛋白质一样或类似。

6.酵母菌可表达外源蛋白与末端前导肽融合，指导新生肽分泌，同时在分泌过程中可对表达的蛋白进行糖基化修饰。

7.不会形成不溶性的包涵体，易于分离提纯8.移去起始甲硫氨酸，避免了在作为药物中使用中引起免疫反应的问题。

9.酵母菌（主要是酿酒酵母）已完成全基因组测序，他具有比大肠杆菌更完备的基因表达控制机制和对表达产物的加工修饰和分泌能力。

10.酵母可进行蛋白的N-乙酰化，C-甲基化，对定向到膜的胞内表达蛋白具有重要意义。

构建基因工程菌是一个复杂、繁琐的过程，因此构建酵母基因要注意：1、结构简单，易于研究2、繁殖能力强，数目多3、成本低，易于培养、4易于观察。

一．酵母基因工程菌的构建过程：1.目的基因的获取：获取目的基因是实施基因工程的第一步，有三种方法提取目的基因。

（1）从自然界中已有的物种中分离出来：.从基因文库中获取目的基因（俗称：鸟枪法）：将含有某种生物的许多DNA片段，导入受体菌的群体中储存，各个受体菌分别含有这种生物不同的基因，称为基因文库。

参考文献书写规范

参考文献书写规范一、学术文章的引用1．人名（1）“姓”：全部保留；（2）“名”：缩写，只取第一字母。

“名”具有多个单词的，取第一字母后按顺序排列；（3）“姓”在前，“名”在后。

（中文期刊人名按中国习惯，不变动。

若译成英文按本规则处理）；（4）人名之间以”,”分开（注意：是单字符的”,”，中文期刊人名也是如此处理）；（5）最后一位人名以”.”结束（注意：是单字符的”.”，中文期刊人名也是如此处理）。

2．文章名（1）句首第一单词大写；（2）最后以”.”结束（注意：是单字符的”.”中文期刊也是如此处理）。

3．刊物名（1）期刊缩名，英文期刊缩名参看随发的附件；（2）缩名单词之间以单字符的空格隔开；（3）最后”.”结束（注意：是单字符的”.”，中文期刊也是如此处理）；（4）期刊缩名字体为斜体。

（中文期刊也是如此处理）。

4．年：年后以”,”结束。

5．卷（1）“卷”后有时会加“期”，以如下形式表示，“卷(期)”，卷期之间的半括号前不加空格；（2）最后”:”结束（注意：是单字符的”:”）；（3）“卷(期):”全部粗体。

6．页码（1）该文献的起始页–终止页；（2）最后”:”结束（注意：是单字符的”:”中文期刊也是如此处理）。

示例1：K wang H ee Lee, H ai L an Piao,H o-Y oun Kim,S ang M i Choi,Fan Jiang,W olfram Hartung,I ldoo Hwang, J une M. Kwak,I n-J ung Lee, I nhwan Hwang. A ctivation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid. Cell. 2006, 126: 1109–1120,规范化处理后Lee KH,Piao HP,Kim HY,Choi SM,Jiang F,Hartung W, Hwang I, Kwak JM,Lee IJ, Hwang I.A ctivation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid. Cell. 2006, 126: 1109–1120.示例2：蒋向辉, 余显权, 赵福胜, 赵德刚. 苗期特耐冷贵州地方水稻品种孕穗期耐冷性研究. 山地农业生物学报. 2004, 23(4): 288–292.二、著作的引用著者. 书名. 版本(第1版不标注). 出版地: 出版者, 出版年, 起始或起止页码.示例4翟婉明. 车辆-轨道耦合动力学. 北京: 中国铁道出版社. 1997, 74–80.Eisson H N. Immunology: an introduction to molecular and cellular principles of the immune response. 5th ed. New York: Harper and Row, 1974, 3–6.三、文献之间的格式（1）编号：采用阿拉伯数字加”.”的形式；（2）文献以“段落悬挂缩进”的形式书写，悬挂缩进一律0.74 cm。

茶树WRKY19_基因的克隆及其抗寒功能研究

茶叶学报 2024，65 （1）：20−27Acta Tea SinicaDOI：10.20045/ki.issn.2096-0220.2024.01.003引文格式：郭珺珺，伊亮辉，肖清，等. 茶树WRKY19基因的克隆及其抗寒功能研究［J］. 茶叶学报，2024，65（1）：20−27.茶树WRKY19基因的克隆及其抗寒功能研究郭珺珺†，伊亮辉†，肖　清，夏恩华，童　伟*（安徽农业大学茶树生物学与资源利用国家重点实验室，安徽　合肥　230036）摘　要：【目的】茶树作为一种喜温畏寒植物，低温严重制约茶产业的发展。

茶树WRKY19基因的克隆及抗寒功能研究为其抗逆工程育种提供重要理论依据。

【方法】基于前期对茶树WRKY基因家族的鉴定，本研究筛选并克隆得到一个受低温诱导显著上调表达的WRKY转录因子，将其命名为CsWRKY19。

对CsWRKY19进行序列特征分析并结合基因表达抑制试验初步研究其在茶树抗寒中的功能。

【结果】CsWRKY19基因 CDS全长为1 680 bp，编码559个氨基酸；包含2个典型的WRKYGQK保守基序；定量PCR分析表明CsWRKY19表达具有组织特异性，在根和幼嫩芽叶中高表达并受冷驯化诱导显著上调表达；经反义寡核苷酸抑制CsWRKY19表达后，茶树叶片在低温下的损伤程度显著加剧、丙二醛含量显著增加、超氧化物歧化酶活性降低，初步证明CsWRKY19在茶树应答低温胁迫中可能具有重要作用。

【结论】本研究鉴定了一个茶树转录因子CsWRKY19，并初步验证了其在茶树应对低温胁迫中的潜在功能。

关键词：茶树；CsWRKY19；低温胁迫；表达分析；功能研究中图分类号：文献标志码：A文章编号：2096−0220（2024）01−0020−08Identification of CsWRKY19 Involving Cold Tolerance of Tea PlantsGUO Jun-jun† , YI Liang-hui† , XIAO Qing , XIA En-hua , TONG Wei*(State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui 230036, China) Abstract：【Objective】CsWRKY19 in Camellia sinensis was cloned to study its relationship with the adaptation and survival of the normally cold sensitive plant exposed to low temperatures. 【Method】Based upon previously identified WRKY family in tea plants, CsWRKY19showing significant upregulation by low temperatures was screened and cloned.Sequence and expressions of the gene in different tissues under cold stress were determined. By suppressing the expression, the ability of a tea plant to tolerate low temperatures was examined for verification. 【Result】CsWRKY19 had a full CDS of 1,680 bp encoding 559 amino acids and containing 2 typical WRKYGQK conserved motifs. The tissue-specific expression of the gene was relatively high in the buds, young leaves, and roots of a plant. Exposing the plant to low temperatures significantly upregulated the gene expression, and if the expression was suppressed at the same time, worse damage on the leaves resulted. Meanwhile, as the surrounding temperature was lowered, the malondialdehyde content rose significantly and the superoxide dismutase activity declined in the plant. 【Conclusion】The transcription收稿日期：2024−01−03初稿；2024−02−20修改稿基金项目：安徽农业大学大学生创新创业训练计划项目（X202210364672）；安徽省高等学校科学研究项目（自然科学类）重点项目（2022AH050867）。

大肠杆菌中细胞信号传导途径的研究

大肠杆菌中细胞信号传导途径的研究大肠杆菌（Escherichia coli）是一种常见的肠道菌，在人体和动物肠道中都广泛存在。

在人体肠道中，它起到了一定的正常生理功能，但当该菌进入到不适当的环境时，会引起肠胃道疾病，例如腹泻、肠炎等。

大肠杆菌是一种典型的原核生物，其细胞结构相对较简单，但是其生命活动却是非常复杂的。

细胞信号传导途径是大肠杆菌调节细胞内和细胞间通讯的关键过程。

现今，人们已经证实了大肠杆菌中多种不同的信号传导途径，这为我们了解大肠杆菌的生长、繁殖和生存等方面提供了基础。

一、普通细菌信号传导通路普通细菌信号传导通路（Bacterial Common Signaling Pathways）是一种中央代谢信号通路，它参与了大肠杆菌的许多基本生命活动。

在这个通路中，许多生化步骤的前后顺序都具有关键性作用，比如说确定左旋乳酸和右旋乳酸的生成方向、调节中间代谢物的合成等。

通过这个通路的调节，大肠杆菌可以调整自身的代谢活动，适应不同的生长环境。

此外，普通细菌信号传导通路也参与了细胞生长、细胞分裂等生命活动。

二、环境应激信号传导途径环境应激信号传导途径（Environmental Stress Signaling Pathways）是大肠杆菌参与环境应激响应的一个重要途径。

该途径可以感知到常见的环境应激因素，如低温、高温、高盐、酸碱等。

通过感知环境应激因素，大肠杆菌可以调整自身的生命活动和代谢行为，以适应不同的环境条件。

此外，环境应激信号传导途径还具有对细胞凋亡的调节作用。

三、A-1型系统A-1型系统（Type A-1 System）是一种较为特殊的细胞信号传导途径。

在这个途径中，大肠杆菌感知到外源DNA后，会利用一种重要的蛋白酶来降解这些DNA，从而防止其被吸收到细胞内部。

因此，A-1型系统对细胞避免被外源细胞病毒侵入有着重要的作用。

四、基因调控途径大肠杆菌基因调控途径（Gene Regulation）是一种常见的信号传导途径。

植物应答低温胁迫的转录调控网络研究进展

植物应答低温胁迫的转录调控网络研究进展一、本文概述Overview of this article随着全球气候变化的加剧，低温胁迫对植物生长发育和产量形成的影响日益显著。

植物在面对低温胁迫时，通过复杂的转录调控网络来适应和抵抗这一环境压力。

近年来，随着分子生物学、基因组学和转录组学等技术的发展，对植物应答低温胁迫的转录调控网络研究取得了显著的进展。

本文旨在综述当前植物应答低温胁迫的转录调控网络研究的最新进展，包括低温胁迫对植物转录组的影响、关键转录因子及其调控机制、以及转录后调控等方面的内容。

通过对这些研究成果的梳理和分析，有助于深入理解植物低温胁迫应答的分子机制，为植物抗寒育种和农业生产的可持续发展提供理论支撑和实践指导。

With the intensification of global climate change, the impact of low temperature stress on plant growth, development, and yield formation is becoming increasingly significant. Plants adapt and resist low temperature stress through a complex transcriptional regulatory network. In recent years, with the development of molecular biology, genomics, andtranscriptomics technologies, significant progress has been made in the study of transcriptional regulatory networks in response to low temperature stress in plants. This article aims to review the latest progress in the research of transcriptional regulation network of plants responding to low temperature stress, including the effects of low temperature stress on plant transcriptome, key transcription factors and their regulation mechanisms, and post transcriptional regulation. Through the sorting and analysis of these research results, it is helpful to deeply understand the molecular mechanisms of plant response to low temperature stress, and provide theoretical support and practical guidance for plant cold resistance breeding and sustainable development of agricultural production.二、低温胁迫下植物的转录调控机制The transcriptional regulation mechanism of plants under low temperature stress低温胁迫是植物在生长过程中经常面临的一种环境压力，它会对植物的生长、发育和代谢产生深远影响。

基因上游转录因子的预测的步骤总结

基因上游转录因子的预测的步骤总结基因上游序列是转录因子结合的关键区域，预测上游转录因子可以揭示基因的调控机制。

The upstream sequence of a gene is a critical region for transcription factor binding, and predicting upstream transcription factors can reveal the regulatory mechanisms of a gene.第一步是收集目标基因的上游序列，通常包括启动子区域。

The first step is to collect the upstream sequence of the target gene, which typically includes the promoter region.接下来，使用生物信息学工具对上游序列进行分析，识别潜在的转录因子结合位点。

Next, use bioinformatics tools to analyze the upstream sequence and identify potential transcription factor binding sites.可以利用计算工具进行转录因子结合位点的预测，例如基于DNA 序列的预测算法。

Computational tools can be used forpredicting transcription factor binding sites, such as algorithms based on DNA sequences.另外，还可以通过实验验证来确定上游序列中的转录因子结合位点。

Additionally, experimental validation can be used to determine transcription factor binding sites within the upstream sequence.结合转录因子数据库的信息，对预测结果进行进一步筛选和分析。

浅谈低温胁迫对植物的影响

低温胁迫对植物的影响杨万坤 114120238（云南师范大学生命科学学院 11应用生物教育A班）摘要：当环境温度持续低于植物正常所需温度（生物学零度）时，温度对植物形成低温胁迫，对植物的生长、发育和生存造成严重影响。

植物遭受低温逆境胁迫时，从感受低温信号到发生一系列生理生化反应和调节基因表达，进而产生抗寒能力。

研究低温胁迫对植物生长发育、生理生化指标、低温反应基因的表达与调控，对于我们生产生活有着重要意义。

Effect of low temperature stress on plant Abstract:When the environment temperature is consistently lower than the temperature normally required for plants (biological zero)，The temperature of low temperature stress on the formation of the plant, the plant growth, development and survival of a serious impact.Plants under low temperature stress, low temperature signal from the feeling to have a series of physiological and biochemical reactions and the regulation of gene expression, resulting in cold hardiness。

Study of low temperature stress on plant growth, physiological and biochemical indicators of low temperature responsive gene expression and regulation, for our production and life of great significance.关键字：低温胁迫、抗寒性、生理生化指标、基因的表达引言：低温胁迫是影响植物生长、发育和地理分布的重要环境限制因素之一。

冷胁迫响应玉米miRNA的靶基因预测及RACE鉴定[毕业作品]

冷胁迫响应玉米miRNA的靶基因预测及RACE鉴定摘要玉米（Zea mays L.）为禾本科，玉蜀黍属一年生草本C4植物，起源于热带或亚热带地区。

玉米用途十分广泛，是重要的饲料作物，又是食品、化工、燃料及医药等行业的重要原料，因此全球玉米种植面积居粮食作物首位，我国的玉米种植面积也于2008年超过水稻成为第一大粮食作物。

玉米在我国国家粮食安全中起着极其重要的作用，生产生活中对玉米的需求不断增加，因而向高纬度、高海拔地区拓展种植区域的需求不断增加。

但是起源于热带、亚热带地区的玉米，在生长周期中表现出对低温冷害较为敏感，在整个生长周期过程中极易遭受低温冷害的影响而导致减产。

研究表明，逆境胁迫不仅会诱导植物蛋白质编码基因的表达，也会诱导一些非蛋白质编码基因的表达，这类非蛋白质编码基因的表达产物在植物的生长、发育和应对逆境胁迫等过程中发挥重要的调控作用。

microRNA（miRNA）是一类内源性小分子的非编码RNA，它通过对其靶基因mRNA的降解或抑制翻译等进行基因表达调控，进而参与调控植物相关生理活动。

miRNA在植物体的新陈代谢、组织器官发育以及分化中起着重要的作用，同时可以响应多种胁迫，能够在植物感受逆境胁迫过程中，做出相应的适应性调整，在植物抵御逆境胁迫过程屮发挥着非常重要的作用。

本次研究主要是对冷胁迫响应玉米miRNA的靶基因预测与5’RACE鉴定。

通过对玉米三叶一心期幼苗不同处理，提取玉米幼苗二、三叶总RNA，并对提取的总RNA进行反转录、巢式PCR、构建载体转化、培养大肠杆菌等，获得单菌落并测序对比，最终得到玉米中miR171n-3p的剪切产物和剪切位点。

关键词：玉米；冷胁迫；microRNA；RACE；靶基因AbstractMaize （Zea mays L.）is a C4 plant species of gramineous and originated in tropical or subtropical regions.Growth and productivity of maize are severely constrained by chilling stress. Because maize is widely used as forage crop, and is an important raw material for chemical, food, fuel and medicine industry, the planting area of maize ranks first world wide, and it became the first major grain crops in China in 2008. Maize plays an extremely important role in the national food security in our country. The boosting requirement for maize demand of expanding the planting area to the high latitude and high altitude region is increasing. As originated in tropical and subtropical areas, maize showed sensitive to low temperature during the entire growth season.Studies have shown that stress can not only induce the expression of plant protein coding genes, but also induce the expression of some nonprotein-encoding genes.The expression products of these nonprotein-encoding genes play an important regulatory role in the process of plant growth, development and stress. MicroRNA （miRNA）is a kind of non-coding RNA of endogenous small molecule. It is involved in the regulation of plant-related physiological activities by regulating gene expression of its target gene mRNA or inhibiting translation. MiRNA plays an important role in plant metabolism, tissue development and differentiation, and can respond to a variety of stress.It is necessary to make the appropriate adjustment in the process of plant stress and stress, which plays a very important role in the process of plant resistance to stress.This study was mainly based on the prediction of the target gene of maize miRNA response to cold stress and 5'RACE identification. Through the different treatments of maize clover seedling.Total RNA was extracted from the second and third leaves of maize seedling, and the total RNA extracted was reverse transcribed, nested PCR, vector transformation, culture of Escherichia coli.A single colony was obtained and sequenced. The shear product and cleavage site of miR171n-3p in maize were obtained.Key words: maize; cold stress; microRNA; RACE; target gene目录摘要......................................................................................................... I Abstract...................................................................................................... II 第一章绪论 (1)第1节低温冷害的类型及其对玉米的影响 (1)第2节植物microRNA的研究进展 (2)2.1 植物microRNA的生物合成 (2)2.2 植物microRNA的作用机制 (3)第3节玉米microRNA的研究 (3)第4节植物miRNA的研究方法与技术路线 (5)4.1 植物miRNA的研究方法 (5)4.2 技术路线 (6)第二章自交系B73玉米材料的获取和处理 (7)第1节自交系B73玉米材料的获取 (7)1.1 实验材料及仪器 (7)1.2 玉米材料的种植 (7)第2节自交系B73玉米材料的处理 (7)2.1 实验材料及仪器 (7)2.2玉米材料的处理 (7)第三章B73玉米幼苗叶片总RNA的提取 (8)第1节RNA提取实验器材的除RNase灭菌处理 (8)1.1 实验试剂及仪器 (8)1.2 实验方法 (8)第2节B73玉米幼苗叶片总RNA的提取 (8)2.1 实验材料及仪器 (8)2.2 实验方法 (8)第3节结果与分析 (9)第四章miRNA靶基因预测及RACE验证 (10)第1节靶基因预测 (10)第2节5’RACE验证 (10)2.1 实验材料及仪器 (10)2.2 实验方法 (10)第3节靶基因降解组测序 (13)3.1 电泳回收 (14)3.2 连接转化 (14)3.3 PCR进行菌液检测及测序 (14)第4节结果与分析 (14)4.1 两轮巢式PCR引物设计与产物检测 (14)4.2 降解产物RACE验证 (15)结论 (17)致谢......................................................................... 错误!未定义书签。

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Chapter3Gene Regulation During Cold Stress Acclimation in Plants Viswanathan Chinnusamy,Jian-Kang Zhu,and Ramanjulu SunkarAbstractCold stress adversely affects plant growth and development and thus limits crop productivity.Diverse plant species tolerate cold stress to a varying degree,which depends on reprogramming gene expres-sion to modify their physiology,metabolism,and growth.Cold signal in plants is transmitted to activate CBF-dependent(C-repeat/drought-responsive element binding factor-dependent)and CBF-independent transcriptional pathway,of which CBF-dependent pathway activates CBF regulon. CBF transcription factor genes are induced by the constitutively expressed ICE1(inducer of CBF expression1)by binding to the CBF promoter.ICE1–CBF cold response pathway is conserved in diverse plant species.Transgenic analysis in different plant species revealed that cold tolerance can be signiﬁcantly enhanced by genetic engineering CBF pathway.Posttranscriptional regulation at pre-mRNA processing and export from nucleus plays a role in cold acclimation.Small noncoding RNAs,namely micro-RNAs (miRNAs)and small interfering RNAs(siRNAs),are emerging as key players of posttranscriptional gene silencing.Cold stress-regulated miRNAs have been identiﬁed in Arabidopsis and rice.In this chapter, recent advances on cold stress signaling and tolerance are highlighted.Key words:Cold stress,second messengers,CBF regulon,CBF-independent regulation,ICE1, posttranscriptional gene regulation.1.IntroductionTemperature profoundly inﬂuences the metabolism of organismsand thus is a key factor determining the growing season andgeographical distribution of plants.Cold stress can be classi-ﬁed as chilling(<20◦C)and freezing(<0◦C)stress.Temperateplants have evolved a repertoire of adaptive mechanisms such asseed and bud dormancy,photoperiod sensitivity,vernalization, R.Sunkar(ed.),Plant Stress Tolerance,Methods in Molecular Biology639,DOI10.1007/978-1-60761-702-0_3,©Springer Science+Business Media,LLC20103940Chinnusamy,Zhu,and Sunkarsupercooling(prevention of ice formation in xylem parenchymacells up to homogenous ice nucleation temperature,−40◦C),andcold acclimation.In cold acclimation,plants acquire freezing tol-erance on prior exposure to suboptimal,low,nonfreezing tem-peratures.The molecular basis of cold acclimation and acquiredfreezing tolerance in Arabidopsis and winter cereals has been stud-ied extensively.Plants modify their metabolism and growth toadapt to cold stress by reprogramming gene expression duringcold acclimation(1,2).This chapter brieﬂy covers cold stress sig-naling,transcriptional and posttranscriptional regulation of geneexpression in cold acclimation process,and the genetic engineer-ing of crops with enhanced cold tolerance.2.Cold StressSensingThus far,the identity of stress sensor in plants is unknown.Theﬂuid mosaic physical state of the plasma membrane is vital forthe structure and function of cells,as well as to sense tempera-ture stress.The plasma membrane undergoes phase transitions,from a liquid crystalline to a rigid gel phase at low temperatureand to aﬂuid state at high temperature.Thus,a decrease in tem-perature can rapidly induce membrane rigidity at microdomains.Further,protein folding is inﬂuenced by temperature changes.Temperature-induced changes in the physical state of mem-branes and proteins are expected to change the metabolic reac-tions and thus the metabolite concentrations.Therefore,plantcells can sense cold stress through membrane rigidiﬁcation,pro-tein/nucleic acid conformation,and/or metabolite concentration(a speciﬁc metabolite or redox status).In alfalfa and Brassica napus,cold stress-induced plasmamembrane rigidiﬁcation leads to actin cytoskeletal rearrangement,induction of Ca2+channels,and increased cytosolic Ca2+level.These events induce the expression of cold-responsive(COR)genes and cold acclimation.Further,a membrane rigidiﬁer(DMSO)can induce COR genes even at25◦C,whereas a mem-braneﬂuidizer(benzyl alcohol)prevents COR gene expressioneven at0◦C(3,4).Genetic evidence for plants sensing cold stressthrough membrane rigidiﬁcation is from the study of the fad2mutant impaired in the oleic acid desaturase gene of Arabidop-sis.In wild-type Arabidopsis plants,diacylglycerol(DAG)kinaseis induced at14◦C.The fad2mutant(more saturated mem-brane)and transgenic Arabidopsis overexpressing linoleate desat-urase gene showed the expression of DAG kinase at18and12◦C,respectively(5).Cold Tolerance in Plants41 3.SecondMessengersand SignalingCytosolic Ca2+levels act as second messenger of the cold stresssignal(6).Calcium may be imported into the cell or released fromintracellular calcium stores.Patch-clamp studies of cold-inducedpotential changes of the plasma membrane in Arabidopsis mes-ophyll protoplasts showed the cold-activatedcalcium-permeablechannel involved in the regulation of cytosolic Ca2+signatures(7).Membrane rigidiﬁcation induced cytosolic Ca2+signatures;and COR gene expression was impaired by gadolinium,amechanosensitive Ca2+channel blocker,which suggests theinvolvement of mechanosensitive Ca2+channels in cold acclima-tion(4).Pharmocological studies implicated cyclic ADP-ribose-and inositol-1,4,5-triphosphate(IP3)-activated intracellularcalcium channels in COR gene expression(4).Calcium inﬂuxinto the cell appears to activate phospholipase C(PLC)and D(PLD),which produce IP3and phosphatidic acid,respectively.IP3can further amplify Ca2+signatures by activation of IP3-gatedcalcium channels(8).Genetic analysis revealed that loss-of-function mutants of FIERY1(FRY1)inositol polyphosphate1-phosphatase show signiﬁcantly higher and sustained levelsof IP3instead of the transient increase observed in wild-typeplants.This situation leads to higher induction of COR genesand CBFs,the upstream transcription factors(9).In addition,the ca lcium e x changer1(cax1)mutant of Arabidopsis,which isdefective in a vacuolar Ca2+/H+antiporter,exhibited enhancedexpression of C-repeat binding factor/dehydration responsiveelement binding(CBF/DREB)proteins and their target CORgenes(10).Therefore,cytosolic Ca2+signatures are upstream ofthe expression of CBFs and COR genes in cold stress signaling.Cold acclimation induces accumulation of ROS such asH2O2,both in chilling-tolerant Arabidopsis and chilling-sensitivemaize plants.ROS can act as a signaling molecule to reprogramtranscriptome probably through induction of Ca2+signatures andactivation of mitogen-activated protein kinases(MAPKs)(11)and redox-responsive transcription factors.Arabidopsis frostbite1(fro1)mutant,which is defective in the mitochondrial Fe-S sub-unit of complex I(NADH dehydrogenase)of the electron trans-fer chain,shows a constitutively high accumulation of ROS.This high accumulation of ROS in fro1results in reduced CORgene expression and hypersensitivity to freezing stress,probablybecause of desensitization of cells by the constitutively high ROSexpression(12).Cold stress-induced second messenger signatures can bedecoded by different pathways.Calcium signatures are sensedby calcium sensor family proteins,namely calcium-dependent42Chinnusamy,Zhu,and Sunkarprotein kinases(CDPKs),calmodulins(CaMs),and salt overlysensitive3-like(SOS3-like)or calcineurin B-like(CBL)pro-teins.In a transient expression system in maize leaf protoplast,aconstitutively active form of an Arabidopsis CDPK(AtCDPK1)activated the expression of barley HV A1ABA-responsive pro-moter::LUC reporter gene suggesting that AtCDPK is a posi-tive regulator in stress-induced gene transcription(13).Geneticand transgenic analyses implicated CDPKs as positive regulators,but a calmodulin,a SOS3-like or a CBL calcium binding protein,and a protein phosphatase2C(AtPP2CA)are negative regulatorsof gene expression and cold tolerance in ponents ofMAPK cascades are induced or activated by cold and other abi-otic stresses.Genetic and transgenic analyses showed that MAPKsact as a converging point in abiotic stress signaling.ROS accu-mulation under these stresses might be sensed through a MAPKcascade(14).ROS activates the AtMEKK1/ANP1(MAPKKK)–AtMKK2(MAPKK)–AtMPK4/6(MAPK)MAPK cascade,whichpositively regulates cold acclimation in plants(11).Many of thesephosphorylated proteins show activation or induction of geneexpression under multiple stress conditions,and genetic modi-ﬁcation results in alteration of multiple stress responses.Theseresults suggest that the proteins act as connecting nodes of stresssignal networks.Identiﬁcation of the target proteins or transcrip-tion factors of protein kinase or phosphatase cascades will shedfurther light on stress signaling.4.TranscriptionalRegulationChilling-tolerant plants reprogram their transcriptome inresponse to acclimation temperature.Cold-regulated genes con-stitute about4–20%of the genome in Arabidopsis(15).Thepromoter region of many COR genes of Arabidopsis con-tains C-repeat(CRT)/DREs,initially identiﬁed in the promoterof r esponsive to d ehydration29A(RD29A/COR78/LTI78).As well,ABA-responsive elements are present in many cold-induced genes.Genetic screens using dehydration and cold stress-responsive promoter-driven LUCIFERASE(RD29A::LUC andCBF3::LUC)led to the isolation of mutants,which unraveledcold-responsive transcriptional networks.4.1.CBF Regulons and Cold Tolerance Yeast one-hybrid screens to identify CRT/DRE binding pro-teins led to the identiﬁcation of CRT/DREBs(CBFs/DREBs) in Arabidopsis.CBFs belong to the ethylene-responsive element binding factor/APETALA2(ERF/AP2)-type transcription factor family.Arabidopsis encodes three CBF genes(CBF1/DREB1B, CBF2/DREB1C,and CBF3/DREB1A),which are induced within a short period of exposure to cold stress.CBFs bind toCold Tolerance in Plants43CRT/DRE cis-elements in the promoters of COR genes andinduce their expression(16,17).Ectopic expression of CBFsin transgenic Arabidopsis induced the expression of COR genesat warm temperatures and induced constitutive freezing toler-ance.These transgenic Arabidopsis plants were also tolerant tosalt and drought stresses(17–19).Microarray analysis of CBF-overexpressing transgenic plants identiﬁed several CBF targetgenes involved in signaling,transcription,osmolyte biosynthesis,ROS detoxiﬁcation,membrane transport,hormone metabolism,and stress response(20,21).Transgenic overexpression of Ara-bidopsis CBFs is sufﬁcient to induce cold tolerance in diverse plantspecies(Table3.1).Further,CBF homologs have been identiﬁed Table3.1Abiotic stress tolerance of transgenic plants overexpressing CBFsGene Transgenicplant Stress tolerance of transgenic plants ReferencesAtCBF1/2/3Brassicanapus Constitutive overexpression enhanced both basaland acquired freezing tolerance(22)AtCBF1Tomato Constitutive overexpression enhanced oxidativestress tolerance under chilling stress;enhancedtolerance to water-deﬁcit stress(23,24)AtDREB1A/ CBF3Tobacco Transgenic plants expressing RD29A::DREB1Aexhibited enhanced chilling and droughttolerance(25)AtDREB1A/ CBF3Wheat Transgenic plants expressing RD29A pro-moter::AtDREB1A gene showed delayedwater stress symptoms(26)AtCBF3Rice Constitutive overexpression resulted in enhancedtolerance to drought and high salinity and amarginal increase in chilling tolerance(27)AtDREB1A/ CBF3Maize RD29A::CBF3transgenic plants are more toler-ant to cold,drought,and salinity(28)AtCBF1Potato Constitutive or stress-inducible expression ofCBF1or CBF3but not CBF2conferredimproved freezing tolerance to frost-sensitiveSolanum tuberosum(29)OsDREB1Arabidopsis Overexpression in Arabidopsis induced targetCOR genes and conferred enhanced toleranceto freezing and drought stresses(30)OsDREB1A/B Rice Constitutive expression conferred improved tol-erance to cold,drought,and salinity(31)ZmDREB1Arabidopsis Overexpression in Arabidopsis induced CORgenes and conferred tolerance to freezing anddrought(32)BnCBF5and BnCBF17B.napus Overexpression led to increased constitutivefreezing tolerance,increased photochemicalefﬁciency and photosynthetic capacity(33)44Chinnusamy,Zhu,and Sunkarfrom several chilling-tolerant and chilling-sensitive plant speciesand transgenic analysis conﬁrmed their pivotal role in cold accli-mation(Table3.1).These evidences suggest that a CBF transcription networkplays a pivotal role in cold acclimation of evolutionarily diverseplant species.Transcriptome analysis of transgenic tomato andArabidopsis plants overexpressing LeCBF1and AtCBF3revealedthat CBF regulons from freezing-tolerant and freezing-sensitiveplant species differ signiﬁcantly(35).Constitutive overexpression of CBFs under the transcriptionalcontrol of the35S cauliﬂower mosaic virus promoter in transgenicplants resulted in severe growth retardation under normal growthconditions in diverse plant species such as Arabidopsis(18,19,34,36),B.napus(22),tomato(23,24),potato(29),and rice(31).Inhibition of metabolism and change in growth-regulatinghormones appears to be important causes of the growth inhibi-tion of CBF-overexpressing plants.Reduction in the expressionof photosynthetic genes appears to reduce photosynthesis andgrowth under cold stress.Transgenic plants constitutively overex-pressing CBFs showed higher induction of the STZ/ZAT10zincﬁnger transcription factor gene,which appears to repress genesinvolved in photosynthesis and carbohydrate metabolism and thusreduce the growth of these transgenic plants(21).Microarrayanalysis revealed that cold stress regulates several genes involvedin biosynthesis or signaling of hormones such as ABA,gib-berellic acid(GA),and auxin,which suggests the importanceof these hormones in coordinated regulation of cold toleranceand plant development(15).GA promotes important processesin plant growth and development,such as seed germination,growth through elongation,andﬂoral transition.Growth retar-dation of transgenictomato plants constitutively overexpressingAtCBF1was reversed by GA3treatment(24).Thisﬁnding sug-gested a link between CBFs and GA in cold stress-induced growthretardation.During cold stress,growth retardation appears to beregulated by CBFs through nuclear-localized DELLA proteins,which repress growth in Arabidopsis.GA stimulates the degrada-tion of DELLA proteins and promotes growth.CBFs enhancethe expression of GA-inactivating GA2-oxidases,and thus allowthe accumulation of the DELLA protein repressor of GA1-like3(RGL3),which leads to dwarﬁsm and lateﬂowering.Further,mutant plants of DELLA genes encoding GA-insensitive[GAI]repressor of GA1-3[RGA]were signiﬁcantly less freezing toler-ant than were wild-type plants after cold acclimation.Thisﬁnd-ing suggests that DELLAs might contribute signiﬁcantly to coldacclimation and freezing tolerance(37).4.2.Regulators of CBF Expression Transcription of CBF genes is induced by cold stress.Hence, constitutive transcription factors present in the cell at normalCold Tolerance in Plants45 growth temperatures may induce the expression of CBFs on acti-vation by cold stress.A systematic genetic analysis by CBF3::LUC bioluminescent genetic screening led to the identiﬁcation of a constitutively expressed and nuclear-localized transcription fac-tor,i nducer of C BF e xpression1(ICE1)in Arabidopsis.ICE1 encodes a MYC-type basic helix-loop-helix(bHLH)transcription factor,can bind to MYC recognition elements in the CBF3pro-moter,and induces the expression of CBF3during cold acclima-tion.The ice1mutant is defective in both chilling and freezing tolerance,whereas transgenic Arabidopsis overexpressing ICE1 showed enhanced freezing tolerance(38).Transcriptome anal-ysis revealed the dominant ice1mutant with impaired expres-sion of about40%of cold-regulated genes,in particular46%of cold-regulated transcription factor genes(15).Therefore,ICE1 is a master regulator that controls CBF and many other cold-responsive regulons.Overexpression analysis showed that ICE2 (At1g12860,a homolog of ICE1)induces the expression of CBF1and confers enhanced freezing tolerance in Arabidop-sis after cold acclimation(39).In wheat,the ICE1homologs TaICE141and TaICE187are constitutively expressed and acti-vate the wheat CBF group IV,which are associated with freezing tolerance.Overexpression of TaICE141and TaICE187in Ara-bidopsis enhanced CBF and COR gene expression and enhanced freezing tolerance only after cold acclimation.Thisﬁnding sug-gests that similar to Arabidopsis ICE1,wheat ICE1also needs to be activated by cold acclimation(40).ICE1appears to negatively regulate the expression of MYB15(an R2R3-MYB family protein)in Arabidopsis.MYB15 is an upstream transcription factor that negatively regulates CBF expression.Transgenic Arabidopsis overexpressing MYB15 showed reduced expression of CBFs and freezingtolerance, whereas myb15T-DNA knockout mutants showed enhanced cold induction of CBFs and enhanced freezing tolerance.In a yeast two-hybrid system,ICE1interacted with MYB15(41). Further,the expression of MYB15is increased in ice1mutants (R236H and K393R)(41,42).Thus,the ICE1–MYB15inter-action appears to play a role in regulating CBF expression levels during cold acclimation(41).Although ICE1is expressed constitutively,only on expo-sure to low temperature does it induce transcription of the CBF and other cold stress-responsive genes(38,40).Posttranslational modiﬁcations play a key role in regulating the activity of ICE1 under cold stress.Cold stress activates ICE1sumoylation(42) and negatively regulates ICE1levels by targeted proteolysis(43). The Arabidopsis High expression of Osmotically responsive gene1 (HOS1)encodes a RINGﬁnger ubiquitin E3ligase.The nuclear localization of HOS1is enhanced by cold stress.HOS1physically interacts with ICE1and targets ICE1for polyubiquitination and46Chinnusamy,Zhu,and Sunkarproteolysis of ICE1after12h of cold stress.Overexpression ofHOS1in transgenic Arabidopsis results in a substantial reductionin level of ICE1protein and that of its target genes,as well ashypersensitivity to freezing stress.Thus,HOS1mediates ubiqui-tination of ICE1and plays a critical role in maintaining the levelof ICE1target genes in the cell during cold acclimation(43).Sumoylation of proteins prevents the proteasomal degradation oftarget proteins.The null mutant of Arabidopsis SUMO E3ligase,SAZ1(SAP and Miz1),exhibits reduced cold induction of CBFsand the target COR genes,as well as hypersensitivity to chillingand freezing stresses.SIZ1catalyzes SUMO conjugation to K393of ICE1during cold acclimation and thus reduces polyubiquiti-nation of ICE1.Mutation in a K393residue of ICE1impairs itsactivity(42).Hence,SIZ1-mediated sumoylation facilitates ICE1stability and activity,whereas HOS1mediation reduces ICE1pro-tein levels during cold acclimation.Stomata play a crucial role in regulating photosynthesis andtranspiration.Recently,the scream-D dominant mutant and ice1mutant were found to be the same as R236H,which resultsin constitutive stomatal differentiation in the epidermis,andthe entire epidermis differentiates into stomata.Thus,ICE1isrequired for controlled stomatal development.ICE1protein inter-acts and forms a dimer with other bHLH transcription fac-tors,SPEECHLESS(SPCH),MUTE,and FAMA,which regu-late stomatal development.ICE1may act as a link between theformation of stomata and the plant response to environmentalcues(44).Recently,members of the calmodulin binding transcriptionactivator(CAMTA)family proteins have been identiﬁed as tran-scriptional regulators of CBF2expression.Cold-induced expres-sion of CBF2was considerably lower in camta3mutant as com-pared to WT plants.The CAMTA3protein binds to conservedDNA motifs present in CBF2promoter and regulates CBF2expression.The camta1/camta3double mutant exhibited hyper-sensitivity to freezing stress as compared to WT plants.SinceCAMTA proteins can interact with calmodulins,cold-inducedcalcium signals may regulate CBFs expression through CAMTAproteins(45).4.3.CBF1,CBF2,and CBF3Play Different Roles in Cold Acclimation Microarray analysis revealed that CBFs regulate about12%of the cold-responsive transcriptome.Overexpression of CBFs enhances osmolyte accumulation,reduces growth,and enhances abiotic stress tolerance(Table3.1).Constitutive overexpression stud-ies of transgenic Arabidopsis suggested that CBF1,CBF2,and CBF3have redundant functional activities(36).However,the ice1mutant,impaired mainly in CBF3but not CBF1and CBF2, showed chilling and freezing hypersensitivity(38).Studies of the cbf2T-DNA insertion mutant of Arabidopsis revealed that CBFsCold Tolerance in Plants47 have different functions in cold acclimation.cbf2null mutants showed increased expression of CBF1and CBF3and enhanced tolerance to freezing(with or without cold acclimation),dehy-dration,and salt stresses.Further,CBFs show a temporal differ-ence in expression,with the cold-induced expression of CBF1and CBF3preceding that of CBF2.These results suggest that CBF2 negatively regulates CBF1and CBF3to optimize the expression of downstream target genes(45).In potato(Solanum tubero-sum),overexpression of AtCBF2failed to confer freezing toler-ance(29).Transgenic analysis of CBF1and CBF3RNAi lines revealed that both CBF1and CBF3are required for the full set of CBF regulon expression and freezing tolerance(46).Besides CBF2,the C2H2zincﬁnger transcription factor ZAT12negatively regulates the expression of CBF1,CBF2,and CBF3during cold stress.Arabidopsis transgenic plants overex-pressing ZAT12showed decreased expression of CBFs under cold stress(47).los2mutant plants showed an enhanced and more sus-tained induction of ZAT10/STZ during cold stress and enhanced cold sensitivity.LOS2encodes a bifunctional enolase that nega-tively regulates the expression of ZAT10(48).Transgenic Arabidopsis plants overexpressing AtMKK2 showed constitutive expression of CBF2,which suggests that the CBF2expression is probably positively regulated by a MAPK sig-naling cascade(11).Arabidopsis FIERY2(FRY2),which encodes an RNApolymerase II C-terminal domain(CTD)phosphatase, appears to act as a negative regulator of CBFs and their target COR genes because the fry2mutant showed enhanced expres-sion of CBFs and COR genesunder cold stress and ABA.Since the fry2mutant is hypersensitive to freezing despite enhanced expression of CBFs,FRY2may positively regulate the expression of certain genes critical for freezing tolerance(49).The main-tenance of an optimal level of CBFs at an appropriate time is necessary,because constitutive overexpression affects growth and development signiﬁcantly.Further,CBF expression is under the control of a circadian clock.The maximal cold-induced increase in transcription of CBFs occurs when cold stress is imposed4h after dawn.Transgenic plants overexpressing arrhythmic CCA1 showed no temporal difference in the cold induction of CBF expression(50).4.4.CBF-Independent Regulons Genetic and transgenic analyses revealed that several classes of transcription factors besides CBFs play an important role in cold acclimation.The eskimo1(esk1)mutant of Arabidopsis was iden-tiﬁed through freezing tolerance genetic screening.The esk1 mutant accumulated constitutively high levels of proline and exhibited constitutively freezing tolerance.ESK1is constitutively expressed and encodes the protein domain of unknown function (23).Transcriptome comparison of CBF2-overexpressing plants48Chinnusamy,Zhu,and Sunkarand esk1mutants showed that different sets of genes are regulatedby CBF2and ESK1.However,the mechanism of action of ESK1in freezing tolerance has yet to be revealed(51).P RD29A::LUC reporter gene-based genetic screening led tothe identiﬁcation of two constitutively expressed transcription fac-tors,HOS9(a homeodomain protein)and HOS10(an R2R3-type MYB),which are necessary for cold tolerance in Arabidop-sis.hos9and hos10mutants are less freezing tolerant than wild-type Arabidopsis(52,53).Transcriptome analysis revealed distinctCBF and HOS9regulons(52).HOS10probably regulates ABA-dependent cold acclimation pathways,because HOS10positivelyregulates NCED3(9-cis-epoxycarotenoid dioxygenase)and thusABA accumulation during cold stress(53).Gene expression analysis revealed several transcription fac-tors induced during cold acclimation.Transgenic analysis of cold-inducible transcription factors helped in validation of functions ofsome transcription factors in cold tolerance.Constitutive overex-pression of the soybean C2H2-type zincﬁnger protein SCOF1in Arabidopsis transgenic plants enhanced the expression of CORgenes and conferred constitutive freezing tolerance.SCOF1inter-acts with soybean G-box binding factor1(SGBF1)and mayenhance the DNA binding activity of the SGBF1.SGBF1isinduced by both cold and ABA(54).Overexpression of the cold-regulated rice transcription factors MYB4(an R2R3-type MYB)and OsMYB3R-2(an R1R2R3MYB)enhanced freezing toleranceof Arabidopsis(55,56).Some members of the abiotic,plant hormone,and pathogen-inducible ERF family play a crucial role in abiotic and bioticstress tolerance.The pepper ERF/AP2-type transcription factorCapsicum annuum pathogen and freezing tolerance-related pro-tein1(CaPF1)is induced by cold,osmotic stress,ethylene,and jasmonic acid.Transgenic Arabidopsis overexpressing CaPF1showed induction of pathogen-responsive as well as COR genesand exhibited enhanced tolerance to stress by freezing and topathogens(Pseudomonas syringae pv tomato DC3000)(57).Sim-ilarly,Triticum aestivum ERF1(TaERF1]was induced by cold,drought salinity,ABA,ethylene,salicylic acid,and infection byBlumeria graminis f.sp.Tritici pathogen in wheat.TransgenicArabidopsis overexpressing TaERF1exhibited enhanced toleranceto cold,salt,and drought stresses,as well as pathogens(58).Genes encoding the A-5subgroup AP2domain protein fromPhyscomitrella patens(PpDBF1)(59)and soybean(GmDREB3)(60)are cold induced,and overexpression of these genes con-ferred enhanced cold tolerance.In wheat,w heat l ow-t emperature-i nduced protein19(WLIP19),encoding a basic-region leucine zipper protein,is induced by cold,drought,and ABA.WLIP19activatesthe expression of COR genes in wheat.Transgenic tobaccooverexpressing Wlip19showed signiﬁcant freezing tolerance.WLIP19was found to interact and form a heterodimer withT.aestivum ocs-element b inding f actor1(TaOBF1),a bZIP tran-scription factor(61).The plant-speciﬁc transcription factor NAC(NAM,ATAF,and CUC)family plays a key role in stressresponse.Overexpression of cold stress-inducible rice SNAC2intransgenic rice resulted in high cell membrane stability under coldstress.Microarray analysis showed upregulation of several stress-regulated genes in SNAC2-overexpressing plants(62).Theseresults suggest that several transcriptional networks operate dur-ing cold acclimation and cold stress tolerance of plants.5.Posttranscrip-tional GeneRegulationPosttranscriptional regulation at pre-mRNA processing,mRNAstability,and export from nucleus plays critical roles in cold accli-mation and cold tolerance(2).5.1.Messenger RNA Processing Pre-mRNA processing and exports constitute important mecha-nisms of regulation of gene expression in eukaryotes.Pre-mRNA undergoes various nuclear processes such as the addition of a5 methyl cap and poly(A)tail and intron splicing.Splicing is nec-essary to remove introns and to synthesize translationally com-petent mRNAs.Primary transcripts with more than one intron can undergo alternative splicing to produce functionally differ-ent proteins from a single gene.In plants,about20%of genes undergo alternative splicing.Although most alternative splicing events are uncharacterized in plants,but it appears to play an important role in the regulation of photosynthesis,ﬂowering, grain quality in cereals,and plant defense response.Recent stud-ies have implicated intron splicing in abiotic stress response.In wheat,cold stress induction of two early cold-regulated(e-cor) genes coding for a ribokinase(7H8)and a C3H2C3RINGﬁn-ger protein(6G2)undergo stress-dependent splicing.Both of these genes are regulated by intron retention under cold stress, whereas6G2intron retention is also regulated by drought stress. However,homologs of these genes did not show stress-regulated intron retention in Arabidopsis.Interestingly,barley homologs of 7H8and6G2showed stress-dependent intron retention under cold stress,whereas barley albino mutants defective in chloro-plast development failed to retain introns in these genes under cold stress(63).The Arabidopsis COR15A gene encoding a chloroplast stromal protein with cryoprotective activity plays an important role in conferring freezing tolerance to chloroplasts (64).The Arabidopsis stabilized1(sta1)mutant is defective in the。