蓝光和蔗糖对拟南芥花色素苷积累和CHS基因表达的影响

以在20μmol m^-2s^-1白光下生长13d的拟南芥(Arabidopsis thaliana,Landsbcrg生态型)幼苗为材料,采用测定叶片花色素苷含量和Northern blot方法,研究蓝光与蔗糖在诱导植物花色素苷积累及相关基因表达中的作用。结果表明：蓝光处理后,叶片花色素苷积累随光强和照光时间的延长而增加,突变体hy4叶片的花色素苷含量明显低于野生型(WT),说明隐花色素1(cry1)是蓝光诱导花色素苷积累的主要光受体：WT中苯基苯乙烯酮合酶基因(CHS)的表达受蓝光诱导,处理4h即有表达,8h达到最高,之后逐渐下降;蓝光不能诱导突变体hy4中CHS基因的表达,说明cry1介导蓝光诱导CHS基因的表达。培养基中不含蔗糖,削弱了蓝光诱导的拟南芥叶片花色素苷的积累,CHS基因表达也受到抑制。蔗糖不仅作为碳源参与蓝光诱导的花色素苷积累,还可能作为信号分子参与蓝光诱导的CHS表达。     

转CiCHS基因拟南芥的黄酮代谢及抗氧化能力分析

该研究克隆了中间锦鸡儿的查尔酮合成酶基因(CiCHS)并转入野生型拟南芥和tt4突变体,用qRT-PCR检测了转基因拟南芥中内源AtCHS基因的表达量,用分光光度法分析了转基因拟南芥的总黄酮、丙二醛含量及DPPH自由基清除能力,用HPLC法检测了转基因拟南芥的柚皮苷含量。结果显示:(1)转基因拟南芥中,内源AtCHS基因的表达量约为野生型的十分之一,总黄酮含量明显高于野生型;HPLC测得转基因株系中柚皮苷含量高于野生型;紫外照射处理前后转基因拟南芥中丙二醛积累量明显少于野生型。(2)转基因株系提取物对DPPH自由基清除能力显著高于野生型。(3)CiCHS基因互补拟南芥tt4突变体,转基因株系的种皮呈现浅棕色。研究表明,中间锦鸡儿CiCHS基因异源表达后生成了柚皮苷,使转基因植物的抗氧化性增强,部分恢复了tt4突变体的种皮颜色。     

AtWRKY40参与拟南芥干旱胁迫响应过程

以拟南芥(Arabidopsis thaliana)野生型、AtWRKY40缺失突变体和过表达株系为材料,研究AtWRKY40在植物干旱胁迫响应过程中的作用及其生理和分子机制。结果显示,AtWRKY40受干旱胁迫诱导;AtWRKY40缺失导致干旱胁迫下种子萌发率降低,叶片失水加剧,而AtWRKY40过表达植株呈现出相反的表征;干旱胁迫下,At WRKY40缺失突变体植株叶片过氧化氢(H_2O_2)、超氧阴离子(O_2~-·)及丙二醛(MDA)含量显著高于野生型及其过表达株系,超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性、脯氨酸(Pro)和可溶性糖含量以及相关基因AtCu/ZnSOD、AtCAT1、AtP5C1S、AtG6PD5和AtBAM4表达量显著低于野生型,同时AtWRKY40过表达株系的渗透物质含量和保护酶活性及其基因表达量则高于野生型。由此说明,AtWRKY40通过调节植株抗氧化能力及渗透调节能力参与拟南芥干旱胁迫响应过程。     

4种李属彩叶植物色素含量与叶色参数的关系

以4种李属彩叶植物紫叶李、紫叶矮樱、黑杆樱李和美人梅4年生嫁接苗为试材,对其叶绿素、类胡萝卜素、花色素苷含量及叶色参数(L·、a·、b·)进行了测定分析.结果表明:(1)随着季节的变化, 4种李属彩叶植物色素含量表现出基本相似的波动趋势,并以黑杆樱李的花色素苷含量在季节间较为稳定且极显著高于其他3种植物(P＜0.01),而美人梅的叶绿素、类胡萝卜素含量在在季节间较为稳定且显著低于其余植物(P＜0.05); 4种植物中各色素含量比例不尽相同且随着季节而变化,但黑杆樱李的比值较稳定且花色素苷比例始终最高,叶片保持紫红色,而美人梅的各色素比值变化较大,叶片呈现紫红-绿-红的变化过程.(2)4种李属彩叶植物的叶色参数(L·、a·、b·)也随季节发生波动,并以黑杆樱李相对稳定,美人梅的变化幅度较大,而且各树种之间的a·和b·值差异分别达到显著水平(P＜0.05)和极显著水平(P<0.01).(3)4种植物叶片 a·值与叶绿素含量、花色素苷含量均呈显著或极显著的正相关性,而L·值和b·值与叶绿素含量、花色素苷含量、类胡萝卜素含量的相关性因树种而异.研究发现,4种李属彩叶植物的色素含量及其比值随季节发生波动的幅度不尽相同,叶片花色素苷含量比例的高-低-高变化使叶色呈现红-绿-红的过渡;花色素苷含量和叶绿素含量的高低决定着a·值大小,但它们并不是影响L·和b·值的主要因素.     

胞外钙离子以及细胞膜组分参与蓝光诱导拟南芥叶片花色素苷的积累

以野生型和hy4突变体拟南芥为材料,运用药物学方法研究可能参与蓝光诱导叶片花色素苷积累和CHS基因表达的信号组分。培养基中外施Ca2 、钙离子通道剂A23187、螯合剂EGTA、钙通道阻断剂尼群地平(nifedipine,Nif)以及异博定(verapermil)的实验证实,蓝光诱导13d龄叶片花色素苷积累和CHS基因表达需要胞外Ca2 的参与,而蓝光作用是由cry1(cryptochrome1)介导的。此外,质膜黄素蛋白抑制剂DPI(diphenylene iodonium)抑制蓝光诱导的花色素苷积累,质膜H -ATPase激活剂壳梭胞素(fusicoccin,FC)抑制蓝光反应,而抑制剂钒酸钠则起促进作用。CaM拮抗剂W7、Ca2 -ATPase抑制剂EB(erythrosine B)、G蛋白激活剂霍乱霉素(cholera toxin,CTX)以及抑制剂百日咳毒素(pertussis toxin,PTX)对蓝光下野生型与hy4的花色素苷积累都有影响。对药物实验的分析表明,质膜氧化还原系统、H -ATPase可能参与依赖于外源Ca2 的蓝光反应。     

拟南芥APX家族基因在植物生长发育与非生物逆境胁迫响应中的作用分析

活性氧造成的氧化胁迫是植物主要非生物逆境胁迫之一。在不利的生长条件下,植物细胞内的各种代谢过程不协调可导致过氧化氢(hydrogen peroxide, H_2O_2)含量增加,从而对细胞造成多种威胁和伤害。抗坏血酸过氧化物酶(ascorbate peroxidase, APX)是植物中清除H_2O_2的一种重要酶,拟南芥(Arabidopsis thaliana) APX家族包括8个成员:APX1～APX6、sAPX和tAPX。本研究以拟南芥野生型和突变体为材料,对拟南芥不同发育时期和不同逆境胁迫下的8种APX基因表达模式进行了分析,同时研究了其相应的缺失突变体对盐、干旱和热胁迫的耐受性。mRNA差异表达模式分析显示:在拟南芥生长的第4～8周,APX1表达量最高,APX2表达量最低,APX4、sAPX和tAPX随着生长发育的时间进程表达量逐渐减少,但APX6表达量不断增加;在非生物胁迫下,APX1、APX2和APX6受热胁迫诱导表达明显,sAPX响应盐胁迫,APX3和APX5对盐、干旱和热胁迫均表现出明显的诱导表达应答。盐和干旱胁迫耐受性分析结果表明:无论是在拟南芥的萌发期还是成熟期,任何一个APX基因缺失均使抗逆性降低;在萌发期,与盐胁迫相比,突变体对干旱胁迫更敏感;在成熟期,与野生型和其他突变体相比,apx1和apx6对盐和干旱胁迫更加不耐受。生理指标检测结果显示:干旱胁迫10 d后,所有突变体植株中的H_2O_2含量均明显高于野生型,其中apx1、sapx和tapx中最高;盐胁迫5 d后,突变体中丙二醛(malondialdehyde,MDA)的含量显著高于野生型;热胁迫2h就会导致apx1、apx2和apx6中H_2O_2和MDA含量大幅增加,其中在apx2中最高。本研究结果表明,拟南芥APX基因家族的8个成员均不同程度地参与植物生长发育及非生物胁迫响应的过程,在不同发育时期或逆境响应过程有特定的一种或几种APX发挥主要作用。     

DELLA蛋白缺失对拟南芥干旱胁迫耐受性的影响

为探索DELLA蛋白缺失对拟南芥耐旱能力的影响,对拟南芥野生型Ler和DELLA蛋白缺失突变体della进行干旱处理,测定存活率、萌发率、离体叶片的失水率、脯氨酸、可溶性糖和丙二醛含量,并对发挥植物细胞脱水保护功能的胚胎晚期丰富蛋白编码基因LEA和ABA应答基因LOX3、COR15b、COR413的表达量进行了检测。结果表明:(1)干旱21d后复水,della突变体的存活率明显高于野生型Ler;(2)della突变体在含甘露醇的固体培养基上的萌发率显著高于Ler;(3)della突变体离体叶片的失水速率明显低于Ler;(4)干旱胁迫后,della突变体脯氨酸、可溶性糖和丙二醛含量的积累低于Ler;(5)干旱胁迫后,della突变体的LEA基因上调表达程度高于Ler,而ABA应答基因上调表达程度低于Ler。研究表明,DELLA蛋白的缺失有助于提高植物抗旱能力。     

拟南芥种皮色素形成突变体的筛选与表型鉴定

类黄酮在植物应答各种环境胁迫和种皮发育调控中起着重要作用。通过甲基磺酸乙酯（EMS）诱变筛选获得1个透明种皮突变体,与野生型拟南芥（Arabidopsis thaliana）(Col-0)相比,突变体成熟的种子颜色为黄色,其表型性状由隐性单基因控制。利用图位克隆和精细定位技术将突变基因定位于5号染色体MAH20的BAC上,是TT4(At5G13930)基因的第1 299位碱基C突变为T,使得第324位氨基酸甘氨酸突变为谷氨酸。TT4(transparent testa 4)编码1个类黄酮合成的结构基因查尔酮合酶（CHS）,突变后种皮透明,种子颜色为黄色,突变体命名为tt4-1。利用功能回补突变体恢复褐色种皮表型,进一步证明了TT4在调节种皮颜色发育过程的重要作用。启动子偶联GUS基因组织表达分析显示TT4基因在植株幼苗的根、茎、叶和花中均有表达,生理表型分析结果显示与野生型相比,突变体tt4-1种子萌发早,幼苗主根短、侧根和根毛较多,成苗叶片气孔开度大和失水率高等特性。该研究将为进一步阐述TT4基因功能奠定理论依据。     

干旱胁迫下转ClNAC9基因露地菊品种‘纽9717’叶片光合及叶绿素荧光特性的比较

对干旱胁迫0~15 d露地菊(Chrysanthemum morifolium Ramat.)品种‘纽9717’(‘Niu 9717’)野生型和3个转ClNAC9基因株系(ClNAC9-5、ClNAC9-6和ClNAC9-13株系)叶片的叶绿素含量、光合参数和叶绿素荧光参数进行了比较和分析。结果表明:干旱胁迫0~15 d,野生型和3个转ClNAC9基因株系叶片中叶绿素含量总体上呈先升高后降低的变化趋势。与干旱胁迫0 d相比较,除初始荧光(F_o)和非光化学淬灭系数(qN)外,干旱胁迫15 d时3个转ClNAC9基因株系叶片的叶绿素含量、净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)、水分利用效率(WUE)、最大荧光(F_m)、可变荧光(F_v)、PSⅡ最大光化学效率(F_v/F_m)、PSⅡ潜在光化学活性(F_v/F_o)和光化学淬灭系数(qP)的降幅均小于野生型。总体上看,干旱胁迫15 d,3个转ClNAC9基因株系叶片的叶绿素含量、Pn、Gs、Tr、WUE、F_m、F_v、F_v/F_m、F_v/F_o、qP和qN值显著高于野生型,而F_o值显著低于野生型。研究结果显示:将ClNAC9基因转入露地菊品种‘纽9717’可以增强该品种对干旱胁迫的耐受性,其中,ClNAC9-5和ClNAC9-6株系对干旱胁迫的耐受性较强,ClNAC9-13株系对干旱胁迫的耐受性较弱,但均强于野生型。     

干旱胁迫下H_2S信号增强植物叶片的光合作用

干旱胁迫是影响农作物产量最重要的环境因素之一。硫化氢(H_2S)作为第三种气体信号分子在植物体内具有多样且积极的生理功能。目前已了解,H_2S在响应植物干旱胁迫应答以及增强植物光合作用的过程中发挥重要作用,但关于内源性H_2S对干旱胁迫下植物光合作用的调节机制未见报道。该研究以拟南芥哥伦比亚野生型(wild type Col-0,WT)、H_2S产生酶编码基因DES缺失突变体des以及H_2S产生酶编码基因DES过表达突变体OE-DES为实验材料,研究内源性H_2S对干旱胁迫下拟南芥光合作用的调节机制。研究结果显示,植株在正常生长条件下,内源性H_2S促使叶片净光合速率、蒸腾速率、叶绿素含量显著升高;植株遭受干旱胁迫时,内源性H_2S可以显著上调Rubisco和Rubisco活化酶(Rubisco activase,RCA)的表达水平,保护叶绿体结构的完整性,促使叶片净光合速率显著上升,维持叶片相应的蒸腾速率,并且引起叶片气孔关闭和胞间CO_2浓度显著升高。     

The antioxidative role of anthocyanins in Arabidopsis under high-irradiance

To uncover the potential antioxidative role of anthocyanins in vivo in protecting photosynthetic tissues from photoinhibition, the effects of high irradiance [HI, 1300 μmol(photon) m⁻² s⁻¹] were studied using detached leaves derived from Arabidopsis wild-type (WT) and the mutant deficient in anthocyanin biosynthesis (tt3tt4). HI stress caused decreased chlorophyll content and photochemical efficiency, but increased cell-membrane leakage and contents of hydrogen peroxide and superoxide radical in the leaves of both Arabidopsis phenotypes, but the WT plants showed better HI tolerance than tt3tt4 mutant. HI caused a significant increase in the 1,1-diphenyl-2-picrylhydrazyl scavenging capacity in WT but not in the tt3tt4 mutant. The anthocyanins could not contribute substantially to light-shielding during the periods of HI stress, because the anthocyanin content in WT was very low and the colour of leaves was the same as in the tt3tt4 mutant. Thus, it was assumed that the better HI tolerance in WT was mostly related to the potential antioxidative role of anthocyanins.     

Protective effect of supplemental anthocyanins on Arabidopsis leaves under high light

Ten anthocyanin components have been detected in roots of purple sweet potato (Ipomoea batatas Lam.) by high‐performance liquid chromatography coupled to diode array detection and electrospray ionization tandem mass spectrometry. All the anthocyanins were exclusively cyanidins or peonidin 3‐sophoroside‐5‐glucosides and their acylated derivatives. The total anthocyanin content in purple sweet potato powder obtained by solid‐phase extraction was 66 mg g^?1. A strong capacity of purple sweet potato anthocyanins (PSPA) to scavenge reactive oxygen species (superoxide, hydroxyl radical) and the stable 1,1‐diphenyl‐2‐picrylhydrazyl organic free radical was found in vitro using the electron spin resonance technique. To determine the functional roles of anthocyanins in leaves in vivo, for the first time, supplemental anthocyanins were infiltrated into leaves of Arabidopsis thaliana double mutant of the ecotype Landsberg erecta (tt3tt4) deficient in anthocyanin biosynthesis. Chlorophyll fluorescence imaging showed that anthocyanins significantly ameliorated the inactivation of photosystems II during prolonged high‐light (1300 µmol m^?2 s^?1) exposure. Comet assay of DNA revealed an obvious role of supplemental PSPA in alleviating DNA damage by high light in leaves. Our results suggest that anthocyanins could function in vitro and in vivo to alleviate the direct or indirect oxidative damage of the photosynthetic apparatus and DNA in plants caused by high‐light stress.     

Expression ofCHS, CHI, andDFR genes in response to light in small radish seedlings

The expression patterns of the genes involved in flavonoid biosynthesis and the changes in anthocyanin content were investigated in small radish (Raphanus sativus L. varsativus) seedlings during light treatment. Anthocyanin content increased until day 4, reaching about 100-fold greater than the control plants, then decreased.CHS (chalcone synthase) mRNA reached a maximum level at 4 h, remained at relatively high levels until day 3, and then decreased rapidly. TheCHI (chalcone isomerase) andDFR (dihydrofolate reductase) mRNA levels reached maximum at 6 h and day 2, respectively, but were decreased rapidly thereafter. All the genes were expressed strongly in hypocotyls, but were either expressed weakly in roots or not expressed at all in cotyledons. Genomic hybridization showed that theCHS gene belonged to a small multigene family, while theCHI andDFR genes were present in one copy per haploid genome.     

Identification and expression analysis of chalcone synthase and dihydroflavonol 4-reductase in Clivia miniata

Clivia miniata is a popular breeding variety. The production of anthocyanin has been studied in Clivia species and the presence of key genes in anthocyanin production, chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR) confirmed. However, it is currently unknown to what extent these genes are expressed in different flower tissue during flower development. Thus the aim of this study was to determine the expression of CHS and DFR in C. miniata var. miniata, an orange flowered variety, and C. miniata var. citrina, a yellow flowered variety, in tepal, carpel and stamen at flower developmental stage two to six. As expected, the anthocyanin content in orange flowers was higher than that of yellow flowers. The expression of CHS and DFR correlated to anthocyanin content. Anthocyanin gene expression and production was found primarily in the tepal. There was a high correlation between CHS and DFR expression suggesting that these genes are subject to coordinate regulation in C. miniata.