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。     

AtMES1正调控拟南芥角粒数

基于拟南芥(Arabidopsis thaliana(L.)Heynh)单角果种子数量(角粒数)的全基因组关联分析(GWAS)数据,筛选到一个可能影响角粒数的候选基因AtMES1,并对其表达模式和转录组数据进行了分析。结果显示,AtMES1的表达模式分析结果表明其在心皮和花序处等特异性表达。分析该基因T?DNA插入突变体的表型发现其角粒数比野生型对照显著下降。转录组分析结果表明,参与调控胚珠发生和发育的多个重要基因在缺失突变体中表达下调。同时使用胎座特异启动子使AtMES1在雌蕊中过量表达,发现转基因植株的角果长度变短,但种子密度显著增加,原因可能是通过激活植物激素相关通路,从而调控胚珠发生和发育相关基因表达正调控角粒数。研究结果初步证实了AtMES1具有正调控拟南芥角粒数和种子密度的功能。     

拟南芥AtLH基因过量表达引起叶向下性卷曲和晚开花(英文)

AtLH基因是BcpLH基因在拟南芥(Arabidopsis thealiana L.)中的同源基因,含有两个编码双链RNA结合蛋白的结构域。在大白菜叶球发育过程中,BcpLH基因与包叶的卷曲有关。为研究AtLH基因对叶卷曲这一重要生物学现象的调控作用,构建了35S:AtLH基因的正义表达载体并转化拟南芥。与野生型比较而言,转基因植株的花和叶中AtLH的表达量有显著增加,成为AtLH基因过量表达的植株。这些植株的莲座叶向外或向下卷曲,呈现明显的偏上性生长;而且抽苔和开花时间延迟;在营养生长期其短缩茎的叶腋处着生数个侧茎,表现为顶端优势减弱;在生殖生长期二级花序减少使得主花序更加发达,表现为顶端优势增强;转基因植株对激素的敏感性改变,IAA刺激根生长的作用增强,ABA抑制根生长的作用减弱。由此可见,AtLH基因的过量表达可引起转基因植株的叶片向下卷曲。     

拟南芥转录因子bHLH17负调控茉莉素介导的抗性反应

植物激素茉莉素作为抗性信号调控植物对腐生性病原菌和昆虫的抗性, 作为发育信号调控植物根的生长、雄蕊发育、表皮毛形成和叶片衰老。茉莉素受体COI1识别茉莉素分子, 进而与JAZ蛋白互作并诱导其降解, 继而调控多种茉莉素反应。拟南芥(Arabidopsis thaliana) IIId亚组bHLH转录因子(bHLH3、bHLH13、bHLH14和bHLH17)是JAZ的一类靶蛋白。与野生型相比, IIId亚组bHLH转录因子的单突变体对灰霉菌和甜菜夜蛾的抗性无明显差异, 而四突变体对灰霉菌和甜菜夜蛾的抗性增强。该文通过高表达bHLH17并研究其对灰霉菌和甜菜夜蛾的抗性反应, 结果显示, 被灰霉菌侵染的bHLH17高表达植株较野生型表现出更严重的病症。取食bHLH17高表达植株叶片的甜菜夜蛾幼虫体重大于取食野生型叶片的幼虫体重。bHLH17高表达抑制了茉莉素诱导的抗性相关基因(Thi2.1)和伤害响应基因(VSP2、AOS、JAZ1、JAZ9和JAZ10)的表达。原生质体转化实验显示bHLH17通过其N端行使转录抑制功能。研究结果表明, IIId亚组bHLH转录抑制因子bHLH17高表达会负调控茉莉素介导的对灰霉菌和甜菜夜蛾的抗性。     

拟南芥RPP1A参与幼苗生长的蛋白质组学分析

核糖体蛋白不仅参与蛋白质合成,而且参与植物生长发育的调控.利用拟南芥核糖体磷酸蛋白P1(ribosomal phosphoprotein P1,RPP1)家族基因RPP1A缺失突变体rpp1a研究RPP1A缺失对幼苗蛋白质表达水平的影响,揭示其参与调控幼苗生长的作用机制.表型分析发现,与野生型WT相比,RPP1A缺失导...     

拟南芥ABA敏感突变体asm1的分离与鉴定

以拟南芥(Arabidopsis thaliana)为研究材料,从T-DNA突变体库中筛选分离得到1株脱落酸(ABA)敏感突变体asm1(ABA sensitive mutant 1,asm1),在含有ABA的培养基中,与野生型相比,asm1突变体的根伸长明显受到抑制,且其种子萌发结果显示asm1对ABA同样表现出敏感特性。在生长发育方面,asm1突变体抽苔时间提前,植株矮化,并且荚果长度明显小于野生型。利用远红外成像系统分析发现,在干旱胁迫下asm1突变体叶面温度高于野生型;失水率分析显示突变体失水率降低以及水分散失减少。遗传学分析表明,asm1是单基因隐性突变且与一个T-DNA插入共分离;通过图位克隆成功获得候选基因ASM1。RT-PCR结果显示,在突变体中ASM1的表达受到抑制,并且能够调控多种ABA信号通路和胁迫应答基因的表达水平。研究结果表明,ASM1可能参与调控ABA信号转导并应答干旱胁迫。     

拟南芥IQM1涉及主根生长对茉莉酸甲酯的反应

目的:进一步找出拟南芥钙调素结合蛋白IQM1介导茉莉酸信号转导的证据。方法:比较分析IQM1基因的功能缺失突变体iqm1-1及其野生型幼苗在茉莉酸甲酯(Me JA)处理后的主根长度和JAZ(jasmonate ZIM-domain)家族基因的表达。结果:Me JA抑制iqm1-1及其野生型植株的主根生长,但iqm1-1对Me JA反应的敏感度比野生型弱;与此结果一致,iqm1-1幼苗中几个JAZ基因表达上调。结论:IQM1介导茉莉酸信号转导,参与对植物根生长的调节。     

PEX5参与调控拟南芥根系抗逆性的初步研究

以拟南芥过氧化物酶体生成蛋白突变体pex5和野生型为试验材料,初步研究PEX5对拟南芥根系抗逆性的影响。结果表明,在MS基础培养基中,突变体pex5的根长相较于野生型根长显著变短;同时,拟南芥pex5突变体株高也低于野生型,说明PEX5基因功能缺失影响了拟南芥根系和茎的生长。pex5突变体在添加氯化钠或甘露醇的MS培养基上培养时,萌发和根伸长受到的抑制显著高于野生型,突变体幼苗的SOD和POD活性也显著低于野生型,说明PEX5参与调控拟南芥根系的抗逆性。本研究为解析PEX5在植物抗逆中的调控机制提供了重要参考。     

拟南芥AtLH基因过量表达引起叶向下性卷曲和晚开花

AtLH基因是BcpLH基因在拟南芥(Arapsis thaliana L.)中的同源基因,含有两个编码双链RNA结合蛋白的结构域.在大白菜叶球发育过程中,BcpLH基因与包叶的卷曲有关.为研究AtLH的基因对叶卷曲这一重要生物学现象的调控作用,构建了35S:AtLH基因的正义表达载体并转化拟南芥.与野生型比较页言,转基因植株的花和叶中AtLH的表达量有显著增加,成为AtLH基因过量的植株.这些植株的莲座叶向外或向下卷曲,呈现明显的偏上性生长;而且抽苔和开花时间延迟;在营养生长期其短缩茎的叶腑处着生数个侧茎,表现为顶端优势减弱;在生殖生长期二级花序减少使得主花序更加发达,表现为顶端优势增强,转基因植株对激素的敏感性改变,IAA剌激根生长的作用增强,ABA抑制根生长的作用减弱.由此可见,AtLH基因的过量表达可引起转基因植株的叶片向下卷曲.     

两个推测的染色质重塑腺苷三磷酸酶基因功能冗余地调控拟南芥种子和胚胎发育

种子及胚胎发育是被子植物个体发育的重要起始阶段,相关调控基因的阐明将有助于认识种子及胚胎发育的分子机制。预测同源基因CHR12和CHR23编码拟南芥依赖于ATP的染色质重塑核心组分—-月泉苷三磷酸酶。T-DNA插入缺失突变体鉴定、遗传杂交及转基因实验表明,两个基因同时缺失阻滞种子的正常发育;胚胎发育进程显微观察表明,与野生型及各自单缺失突变体相比,双重缺失突变体的胚胎发育停滞到了心形胚后期或鱼雷胚早期。这表明CHR12和CHR23在拟南芥种子及胚胎发育过程中功能冗余地发挥着重要调控作用。     

The WAG1 and WAG2 protein kinases negatively regulate root waving in Arabidopsis

The WAG1 and WAG2 genes of Arabidopsis thaliana encode protein-serine/threonine kinases that are closely related to PINOID. In order to determine what roles WAG1 and WAG2 play in seedling development, we used a reverse genetics approach to study the wag1, wag2 and wag1/wag2 mutant phenotypes for clues. Although the wag mutants do not contain detectable amounts of the corresponding mRNA, they are wild type in most respects. However, wag1/wag2 double mutants exhibit a pronounced wavy root phenotype when grown vertically on agar plates, a phenotype observed in wild-type plants only on plates inclined to angles less than 90 degrees. The wag1 and wag2 mutants also demonstrate enhanced root waving, but to a lesser extent. Moreover, the double mutant roots are more resistant to the effects of N-1-naphthylphthalamic acid on the inhibition of root curling, raising the possibility that transport of auxin is affected in the wag mutants. Promoter fusions to the gusA reporter gene demonstrate that the WAG promoters are most active in root tips, consistent with the observed phenotypes in the wag mutants.     

Arabidopsis extra-large G proteins (XLGs) regulate root morphogenesis

As in mammalian systems, heterotrimeric G proteins, composed of alpha, beta and gamma subunits, are present in plants and are involved in the regulation of development and cell signaling. Besides the sole prototypical G protein alpha subunit gene, GPA1, the Arabidopsis thaliana genome has three extra-large GTP-binding protein (XLG)-encoding genes: XLG1 (At2g23460), XLG2 (At4g34390) and XLG3 (At1g31930). The C-termini of the XLGs are Galpha domains that are homologous to GPA1, whereas their N-termini each contain a cysteine-rich region and a putative nuclear localization signal (NLS). GFP fusions with each XLG confirmed nuclear localization. All three XLG genes are expressed in essentially all plant organs, with strong expression in vascular tissues, primary root meristems and lateral root primordia. Analysis of single, double and triple T-DNA insertional mutants of the XLG genes revealed redundancy in XLG function. Dark-grown xlg1-1 xlg2-1 xlg3-1 triple mutant plants showed markedly increased primary root length compared with wild-type plants. This phenotype was not observed in dark-grown xlg single mutants, and was suppressed upon complementation of the xlg triple mutant with each XLG. Root cell sizes of the xlg triple mutant and root morphology were highly similar to those of wild-type roots, suggesting that XLGs may regulate cell proliferation. Dark-grown roots of the xlg triple mutants also showed altered sensitivity to sugars, ABA hyposensitivity and ethylene hypersensitivity, whereas seed germination in xlg triple mutants was hypersensitive to osmotic stress and ABA. As plant-specific proteins, regulatory mechanisms of XLGs may differ from those of conventional Galphas.     

Two homologous ATP-binding cassette transporter proteins, AtMDR1 and AtPGP1, regulate Arabidopsis photomorphogenesis and root development by mediating polar auxin transport

Light and auxin control many aspects of plant growth and development in an overlapping manner. We report here functional characterization of two closely related ABC (ATP-binding cassette) transporter genes, AtMDR1 and AtPGP1, in light and auxin responses. We showed that loss-of-function atmdr1 and atpgp1 mutants display hypersensitivity to far-red, red, and blue-light inhibition of hypocotyl elongation, reduced chlorophyll and anthocyanin accumulation, and abnormal expression of several light-responsive genes, including CAB3, RBCS, CHS, and PORA, under both darkness and far-red light conditions. In addition, we showed that the atmdr1-100 and atmdr1-100/atpgp1-100 mutants are defective in multiple aspects of root development, including increased root-growth sensitivity to 1-naphthalene acetic acid (1-NAA), and decreased sensitivity to naphthylphthalamic acid (NPA)-mediated inhibition of root elongation. Consistent with the proposed role of AtMDR1 in basipetal auxin transport, we found that expression of the auxin responsive DR5::GUS reporter gene in the central elongation zone is significantly reduced in the atmdr1-100 mutant roots treated with 1-NAA at the root tips, compared to similarly treated wild-type plants. Moreover, atmdr1-100, atpgp1-100, and their double mutants produced fewer lateral roots, in the presence or absence of 1-NAA or NPA. The atmdr1-100 and atmdr1-100/atpgp1-100 mutants also displayed enhanced root gravitropism. Genetic-epistasis analysis revealed that mutations in phyA largely suppress the randomized-hypocotyl growth and the short-hypocotyl phenotype of the atmdr1-100 mutants under far-red light, suggesting that phyA acts downstream of AtMDR1. Together, our results suggest that AtMDR1 and AtPGP1 regulate Arabidopsis (Arabidopsis thaliana) photomorphogenesis and multiple aspects of root development by mediating polar auxin transport.     

Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interaction

The identification of a family of NAR2-type genes in higher plants showed that there was a homolog in Arabidopsis (Arabidopsis thaliana), AtNAR2.1. These genes encode part of a two-component nitrate high-affinity transport system (HATS). As the Arabidopsis NRT2 gene family of nitrate transporters has been characterized, we tested the idea that AtNAR2.1 and AtNRT2.1 are partners in a two-component HATS. Results using the yeast split-ubiquitin system and Xenopus oocyte expression showed that the two proteins interacted to give a functional HATS. The growth and nitrogen (N) physiology of two Arabidopsis gene knockout mutants, atnrt2.1-1 and atnar2.1-1, one for each partner protein, were compared. Both types of plants had lost HATS activity at 0.2 mm nitrate, but the effect was more severe in atnar2.1-1 plants. The relationship between plant N status and nitrate transporter expression revealed a pattern that was characteristic of N deficiency that was again stronger in atnar2.1-1. Plants resulting from a cross between both mutants (atnrt2.1-1 x atnar2.1-1) showed a phenotype like that of the atnar2.1-1 mutant when grown in 0.5 mm nitrate. Lateral root assays also revealed growth differences between the two mutants, confirming that atnar2.1-1 had a stronger phenotype. To show that the impaired HATS did not result from the decreased expression of AtNRT2.1, we tested if constitutive root expression of a tobacco (Nicotiana plumbaginifolia) gene, NpNRT2.1, previously been shown to complement atnrt2.1-1, can restore HATS to the atnar2.1-1 mutant. These plants did not recover wild-type nitrate HATS. Taken together, these results show that AtNAR2.1 is essential for HATS of nitrate in Arabidopsis.     

OsCSLD1, a cellulose synthase-like D1 gene, is required for root hair morphogenesis in rice

Root hairs are long tubular outgrowths that form on the surface of specialized epidermal cells. They are required for nutrient and water uptake and interact with the soil microflora. Here we show that the Oryza sativa cellulose synthase-like D1 (OsCSLD1) gene is required for root hair development, as rice (Oryza sativa) mutants that lack OsCSLD1 function develop abnormal root hairs. In these mutants, while hair development is initiated normally, the hairs elongate less than the wild-type hairs and they have kinks and swellings along their length. Because the csld1 mutants develop the same density and number of root hairs along their seminal root as the wild-type plants, we propose that OsCSLD1 function is required for hair elongation but not initiation. Both gene trap expression pattern and in situ hybridization analyses indicate that OsCSLD1 is expressed in only root hair cells. Furthermore, OsCSLD1 is the only member of the four rice CSLD genes that shows root-specific expression. Given that the Arabidopsis (Arabidopsis thaliana) gene KOJAK/AtCSLD3 is required for root hair elongation and is expressed in the root hair, it appears that OsCSLD1 may be the functional ortholog of KOJAK/AtCSLD3 and that these two genes represent the root hair-specific members of this family of proteins. Thus, at least part of the mechanism of root hair morphogenesis in Arabidopsis is conserved in rice.     

Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.     

Compensatory expression and substrate inducibility of gamma-glutamyl transferase GGT2 isoform in Arabidopsis thaliana

γ-Glutamyl transferases (GGT; EC 2.3.2.2) are glutathione-degrading enzymes that are represented in Arabidopsis thaliana by a small gene family of four members. Two isoforms, GGT1 and GGT2, are apoplastic, sharing broad similarities in their amino acid sequences, but they are differently expressed in the tissues: GGT1 is expressed in roots, leaves, and siliques, while GGT2 was thought to be expressed only in siliques. It is demonstrated here that GGT2 is also expressed in wild-type roots, albeit in very small amounts. GGT2 expression is enhanced in ggt1 knockout mutants, suggesting a compensatory effect to restore GGT activity in the root apoplast. Supplementation with 100 μM glutathione (GSH) resulted in the up-regulation of GGT2 gene expression in wild-type and ggt1 knockout roots, and of GGT1 gene expression in wild-type roots. Glutathione recovery was hampered by the GGT inhibitor serine/borate, suggesting a major role for apoplastic GGTs in this process. These findings can explain the ability of ggt1 knockout mutants to retrieve exogenously added glutathione from the growth medium.     

Expression pattern of Aux/IAA genes in the iaa3/shy2-1D mutant of Arabidopsis thaliana (L.)

A semi-dominant mutant suppressor of hy2 (shy2-1D) of Arabidopsis thaliana, originally isolated as a photomorphogenesis mutant, shows altered auxin responses. Recent molecular cloning revealed that the SHY2 gene is identical to the IAA3 gene, a member of the primary auxin-response genes designated the Aux/IAA gene family. Because Aux/IAA proteins are reported to interact with auxin response factors, we investigated the pattern of expression of early auxin genes in the iaa3/shy2-1D mutant. RNA hybridization analysis showed that levels of mRNA accumulation of the early genes were reduced dramatically in the iaa3/shy2-1D mutants, although auxin still enhanced gene expression in the iaa3/shy2-1D mutant. Histochemical analysis using a fusion gene of the auxin responsive domain (AuxRD) and the GUS gene showed no IAA-inducible GUS expression in the root elongation zone of the iaa3/shy2-1D mutant. On the other hand, ectopic GUS expression occurred in the hypocotyl, cotyledon, petiole and root vascular tissues in the absence of auxin. These results suggest that IAA3/SHY2 functions both negatively and positively on early auxin gene expression.