拟南芥F-box蛋白FKF1与转录因子FUL互作调控开花研究

F-box蛋白FLAVIN-BINDING KELCH REPEAT F-BOX 1(FKF1)参与调控拟南芥光周期开花,但其分子机制尚不完全清楚。本研究通过体内和体外实验,证明FKF1与转录因子FRUITFULL(FUL)相互作用。qRT-PCR和Western blot结果显示,FKF1正调节FUL的转录水平,但不影响FUL蛋白的稳定性。遗传分析结果显示,35S-FKF1-Myc / ful-8双突变体的开花表型以及开花基因FLOWERING LOCUS T(FT)的转录水平与ful-8突变体相一致。研究结果表明FKF1可通过与FUL互作,在FUL的上游促进FT表达,进而促进开花。     

拟南芥生物钟分子机制研究进展

本文主要概述了目前拟南芥生物钟分子机制的研究进展。生物钟通过调控导引节律的相位来调节植物的生理活动。拟南芥生物钟由CCA1、LHY和TOC1 3个主要基因构成了一个稳定的负反馈环,来调节昼夜节律中各个基因如APRR/TOC1 5重奏的作用, 从而调控昼夜节律的相位。在开花的光周期调控中, 提出了外协和模型, 其中的关键基因是CO , 它与拟南芥的开花时间直接相关。     

拟南芥生物钟分子机制研究进展

本文主要概述了目前拟南芥生物钟分子机制的研究进展.生物钟通过调控导引节律的相位来调节植物的生理活动.拟南芥生物钟由CCAJ、LHy和TOCJ 3个主要基因构成了一个稳定的负反馈环,来调节昼夜节律中各个基因如APRR/TOC15重奏的作用,从而调控昼夜节律的相位.在开花的光周期调控中,提出了外协和模型,其中的关键基因是CO,它与拟南芥的开花时间直接相关.     

受光调节的拟南芥锌指蛋白DBB（DoubleB-Box）亚家族基因的转录表达

整合微阵列数据和采用实时荧光定量PCR分析拟南芥锌指蛋白DBB亚家族中8个基因在不同光周期和不同光质条件下转录表达的结果表明,在长日和短目照条件下该家族中6个基因的转录都具有光周期节律性,并且其中4个基因的表达受光诱导,有1个基因的表达受光抑制,1个基因的表达不受光调节。     

拟南芥F-box蛋白家族的功能研究进展

F-box蛋白是一类含有F-box基序、在泛素介导的蛋白质水解过程中具有底物识别特性的蛋白质。该文对国内外近年来有关F-box家族在拟南芥中的数量、种类以及在生长发育、细胞信号转导、生物及非生物逆境胁迫等多种生理过程中的作用等方面的研究进展进行综述,以期促进该家族基因在拟南芥和其他重要农作物中的功能研究,尽快描绘出该家族在植物中的代谢网络图谱。     

拟南芥AtDAD1 超量表达植株对H2O2抗性的研究

构建拟南芥AtDAD1超量表达载体,以农杆菌介导的方法转化拟南芥哥伦比亚生态型,比较AtDAD1超量表达植株和野生型植株表现型的差异,以及两者对H2O2抗性的不同。实验显示,AtDAD1转基因拟南芥生长较野生型拟南芥更为强壮,对高浓度H2O2有较强的耐受力。测定两者糖含量,发现AtDAD1转基因拟南芥叶片糖的含量明显高于野生型拟南芥叶片。以上结果表明,AtDAD1基因可能参与植物生长发育,并可能在拟南芥抵抗凋亡的过程中发挥重要的作用。     

拟南芥DG1参与叶绿体早期光合蛋白合成功能

拟南芥中已有466个PPR蛋白,已有研究证实许多PPR蛋白参与细胞器基因表达的转录后调节,但大部分PPR蛋白分子作用机制尚不清楚.Delayed greening 1(DG1)是定位于叶绿体中的的PPR蛋白,研究结果证实该蛋白是通过与SIG6因子相互作用降低PEP转录活性从而影响叶绿体早期发育.本研究利用拟南芥Dg1基因功能缺陷型突变体研究了DG1蛋白对光系统蛋白复合体组成及其光转化效率的影响.77K荧光发射光谱分析发现dg1突变体幼叶PSII中电子传递速度明显低于野生型,而成熟叶片与野生型基本一致;蓝绿温和胶分析结果表明:相对于野生型在dg1突变体新生叶中PSII、PS玉及其超聚复合物含量均有不同程度降低;进一步温和胶二向电泳及蛋白免疫印迹分析显示,在dg1突变体新生叶中,由叶绿体编码的光系统蛋白复合物组成亚基含量显著降低,而核编码复合物组成亚基含量与野生型相比没有明显区别.上述实验结果进一步确定了DG1蛋白是通过调控叶绿体编码基因的表达进而调节光系统复合物的生物合成与组装,最终影响拟南芥叶绿体早期发育.因此,我们认为DG1蛋白对于叶绿体发育早期光合蛋白的合成是必需的.     

拟南芥精氨酸甲基转移酶SKB1基因的分离与功能鉴定

以哥伦比亚"生态型(Columbia ecotype)拟南芥(Arabidopsis thaliana)叶片为材料,采用RT-PCR技术,获得了拟南芥精氨酸甲基转移酶(SKB1)基因,该基因全长1 929 bp,将该基因克隆到原核表达载体pET28b上,并转化大肠杆菌BL21,经IPTG诱导表达大量目的蛋白,其表达量占菌体总蛋白的50%以上.通过切胶回收抗原的方法,制备了兔源AtSKB1多克隆抗体,同时构建了SKB1基因的正义表达载体pBI121-35S∷SKB1并转化拟南芥.Western检测结果显示,转基因植株的SKB1表达量显著增加,同时SKB1表达量增加的植株开花时间也明显提前,说明植株的开花时间与SKB1的表达量呈正相关.结果表明,拟南芥SKB1基因的过量表达可引起植株的早花,SKB1参与了植物的开花发育信号通路.     

拟南芥MAX2蛋白功能研究进展

独角金内酯(strigolactone, SLs)是一类新型植物激素,在植物生长发育的进程中发挥多种重要功能,包括调控植物的分枝,促进种子的萌发,以及影响根系建成等。MAX2 (more axillary growth 2)是SL信号传导途径的关键调控因子,位于合成途径基因MAX1、MAX3和MAX4的下游,几乎影响独脚金内酯所控制的所有表型。近年来,MAX2多样化的功能逐步得到揭示,大量数据表明MAX2不仅仅是SL信号的重要组分,同时也参与SL和多种激素信号间的交叉互作,在植物生长发育的各个环节,以及抵御生物和非生物胁迫的反应中都发挥至关重要的作用,但具体调控机制还有待更加深入的研究。对目前已知的MAX2功能进行了总结和阐述,以期为全面揭示MAX2功能及其调控多种激素信号的交叉机制提供理论参考。     

拟南芥NPH3/RPT2-Like (NRL)家族蛋白在向光素信号转导通路中的作用研究进展

向光素PHOT1和PHOT2感受蓝光刺激后发生自磷酸化激活, 调节植物气孔开放、叶绿体运动、叶片伸展和定位以及向光性(包括根的负向光性和下胚轴的向光性)等多种适应性反应。拟南芥(Arabidopsis thaliana) NRL (NPH3/RPT2-Like)家族成员在向光素介导的信号途径中发挥重要作用, 其中NPH3特异调控下胚轴的向光性以及叶片的伸展与定位, RPT2参与调节植物向光性、叶片的伸展与定位以及叶绿体聚光反应等。NCH1是新发现的NRL家族成员, 与RPT2以功能冗余的方式调节叶绿体的聚光反应, 但不调节避光反应。该文主要综述了NRL蛋白家族成员在向光素介导蓝光信号通路中的作用, 并展望了未来的研究方向, 旨在为全面揭示NRL家族成员的功能提供线索。     

Light-dependent suppression of COP1 multimeric complex formation is determined by the blue-light receptor FKF1 in Arabidopsis

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a multifunctional E3 ligase protein with many target proteins, is involved in diverse developmental processes throughout the plant's lifecycle, including seed germination, the regulation of circadian rhythms, photomorphogenesis, and the control of flowering time. To function, COP1 must form multimeric complexes with SUPPRESSOR OF PHYA1 (SPA1), i.e., [(COP1)₂(SPA1)₂] tetramers. We recently reported that the blue-light receptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX1) represses COP1 activity by inhibiting its homodimerization, but it is not yet clear whether FKF1 affects the formation of COP1-containing multimeric complexes. To explore this issue, we performed size exclusion chromatography (SEC) of Arabidopsis thaliana proteins and found that the levels and composition of COP1-containing multimeric complexes varied throughout a 24-h period. The levels of 440–669?kDa complexes were dramatically reduced in the late afternoon compared to the morning and at night in wild-type plants. During the daytime, the levels of these complexes were reduced in FKF1-overexpressing plants but not in fkf1-t, a loss-of-function mutant of FKF1, suggesting that FKF1 is closely associated with the destabilization of COP1 multimeric protein complexes in a light-dependent manner. We also analyzed the SEC patterns of COP1 multimeric complexes in transgenic plants overexpressing mutant COP1 variants, including COP1^L105A (which forms homodimers) and COP1^L170A (which cannot form homodimers), and found that COP1 multimeric complexes were scarce in plants overexpressing COP1^L170A. These results indicate that COP1 homodimers serve as basic building blocks that assemble into COP1 multimeric complexes with diverse target proteins. We propose that light-activated FKF1 inhibits COP1 homodimerization, mainly by destabilizing 440–669?kDa COP1 complexes, resulting in the repression of CONSTANS-degrading COP1 activity in the late afternoon in long days, but not in short days, thereby regulating photoperiodic flowering in Arabidopsis.     

LOV KELCH PROTEIN2 and ZEITLUPE repress Arabidopsis photoperiodic flowering under non-inductive conditions, dependent on FLAVIN-BINDING KELCH REPEAT F-BOX1

LOV KELCH PROTEIN2 (LKP2), ZEITLUPE (ZTL)/LOV KELCH PROTEIN1 (LKP1) and FLAVIN‐BINDING KELCH REPEAT F‐BOX1 (FKF1) constitute a family of Arabidopsis F‐box proteins that regulate the circadian clock. Over‐expression of LKP2 or ZTL causes arrhythmicity of multiple clock outputs under constant light and in constant darkness. Here, we show the significance of LKP2 and ZTL in the photoperiodic control of flowering time in Arabidopsis. In plants over‐expressing LKP2, CO and FT expression was down‐regulated under long‐day conditions. LKP2 and ZTL physically interacted with FKF1, which was recruited from the nucleus into cytosolic speckles. LKP2 and ZTL inhibited the interaction of FKF1 with CYCLING DOF FACTOR 1, a ubiquitination substrate for FKF1 that is localized in the nucleus. The Kelch repeat regions of LKP2 and ZTL were sufficient for their physical interaction with FKF1 and translocation of FKF1 to the cytoplasm. Over‐expression of LKP2 Kelch repeats induced late flowering under long‐day conditions. lkp2 ztl double mutant plants flowered earlier than wild‐type plants under short‐day (non‐inductive) conditions, and both CO and FT expression levels were up‐regulated in the double mutant plants. The early flowering of lkp2 ztl was dependent on FKF1. LKP2, ZTL or both affected the accumulation of FKF1 protein during the early light period. These results indicate that an important role of LKP2 and ZTL in the photoperiodic pathway is repression of flowering under non‐inductive conditions, and this is dependent on FKF1.     

LKP1 (LOV kelch protein 1): a factor involved in the regulation of flowering time in arabidopsis

In plants, light is not only an energy source but also a very important signal that modulates development and differentiation. Here, we report a putative photo-regulatory factor sequence in LKP1 (LOV kelch protein 1). LKP1 cDNA encodes a protein of 610 amino acids and with a molecular weight of 65 905 with an LOV domain and kelch repeats. LOV domains are present in a number of sensor proteins involved in the detection of light, oxygen or voltage. The LKP1 LOV is very similar to the LOV domains in NPH1, a plasma membrane-associated blue light receptor kinase that regulates phototropism (Huala, E., Oeller, P.W., Liscum, E., Han, I-S., Larsen, E. & Briggs, W.R. (1997) Science, 278, 2120-2123). LKP1 mRNA accumulates in roots, stems, flowers and siliques. It is most abundant in leaves, and least abundant in seeds. Transgenic plants with a beta-glucuronidase (GUS) reporter gene driven by a 1.5 kb LKP1 promoter display strong GUS activity in leaves. Transgenic plants with a 35S:LKP1 cDNA gene overexpress LKP1 mRNA. These plants have elongated hypocotyls and petioles with elongated cells, and exhibit distinct cotyledon movement during the day. Expression of 35S:LKP1 in transgenic Arabidopsis promotes late flowering in plants grown under long-day, but not under short-day conditions. Vernalization does not affect the late flowering phenotype of the 35S:LKP1 plants. Transgenic plants possessing the 35S:GFP-LKP1 construct also have long hypocotyles and petioles, and a late flowering phenotype, suggesting that the GFP-LKP1 fusion protein is active. The GFP-associated fluorescence in 35S:GFP-LKP1 plants is observed in nuclei and cytosol, indicating that LKP1 is a new nucleo-cytoplasmic factor that influences flowering time in the long day pathway of Arabidopsis.     

Quaternary structure of LOV-domain containing polypeptide of Arabidopsis FKF1 protein

Flavin-binding, Kelch repeat, F-box (FKF1) protein is a photoreceptor to regulate flowering of Arabidopsis. The protein has a light, oxygen and voltage (LOV)-sensing domain binding a flavin mononucleotide. The photo-activation of the domain is an indispensable step to initiate the cellular signaling for flowering. In the present study, a LOV-containing polypeptide of FKF1 was prepared by an overexpression system, and the quaternary structure of it was studied by size exclusion chromatography and small-angle X-ray scattering. The apparent molecular weight from chromatography suggested a globular trimeric or an anisotropic-shaped dimeric association of the polypeptide in solution. The scattering experiment demonstrated a dimeric association of the polypeptides with an elongated molecular shape displaying the radius of gyration of 27 A and the maximum dimension of 94 A. The molecular shape simulated from scattering profiles suggests an antiparallel association of the LOV domains in the dimer. Though the absorption spectrum of blue-light irradiated polypeptide was stable in the photoactivated state for a long period, the scattering profiles showed very small changes between the dark and light conditions. Based on the homologies in the amino-acid sequences and the scattering profiles, these results are discussed in connection with the structures and function of LOV domains of phototropin.     

Genetic linkages of the circadian clock-associated genes, TOC1, CCA1 and LHY, in the photoperiodic control of flowering time in Arabidopsis thaliana

In Arabidopsis thaliana, the flowering time is regulated through the circadian clock that measures day-length and modulates the photoperiodic CO-FT output pathway in accordance with the external coincidence model. Nevertheless, the genetic linkages between the major clock-associated TOC1, CCA1 and LHY genes and the canonical CO-FT flowering pathway are less clear. By employing a set of mutants including an extremely early flowering toc1 cca1 lhy triple mutant, here we showed that CCA1 and LHY act redundantly as negative regulators of the photoperiodic flowering pathway. The partly redundant CCA1/LHY functions are largely, but not absolutely, dependent on the upstream TOC1 gene that serves as an activator. The results of examination with reference to the expression profiles of CO and FT in the mutants indicated that this clock circuitry is indeed linked to the CO-FT output pathway, if not exclusively. For this linkage, the phase control of certain flowering-associated genes, GI, CDF1 and FKF1, appears to be crucial. Furthermore, the genetic linkage between TOC1 and CCA1/LHY is compatible with the negative and positive feedback loop, which is currently believed to be a core of the circadian clock. The results of this study suggested that the circadian clock might open an exit for a photoperiodic output pathway during the daytime. In the context of the current clock model, these results will be discussed in connection with the previous finding that the same clock might open an exit for the early photomorphogenic output pathway during the night-time.     

Photoperiod-regulated expression of the PpCOL1 gene encoding a homolog of CO/COL proteins in the moss Physcomitrella patens

The CONSTANS (CO) protein is a critical regulator of the photoperiodic control of flowering in Arabidopsis thaliana and Oryza sativa. We isolated a cDNA PpCOL1 encoding a homolog of the CO/CO-LIKE (COL) family proteins from a cryptogam Physcomitrella patens. The predicted PpCOL1 protein has N-terminal zinc finger and C-terminal CCT domains, which are conserved in the angiosperm CO/COL proteins. Structurally, PpCOL1 is the most closely related to the Group Ia or Ic proteins, which include AtCO and AtCOL1/2, among diverged members of the family. A transient expression assay using GFP showed that the CCT domain of PpCOL1 contains a nuclear-localizing signal. Northern blotting analyses revealed that the PpCOL1 expression is controlled by the circadian clock, and moreover, it is photoperiodically regulated at a gametophore stage when the rate of sporophyte formation is affected by day length. These observations indicate a possible involvement of PpCOL1 as a nuclear factor in the photoperiodic regulation of reproduction of Physcomitrella.     

Analysis of leaf proteins in late flowering mutants of Arabidopsis thaliana

Late flowering monogenic mutants of Arabidopsis thaliana (L.) Heynh. at the loci co, gi, fca, fve, fwa, fha, fpa, fy and their corresponding wild type, Landsberg erecta , were analysed by two-dimensional gel electrophoresis. All plants were grown under continuous light and proteins were extracted from leaves of the same age (20-day-old). The polypeptide patterns of the mutants at the loci co, gi, fca, fve, fwa, fha, fpa , and Landsberg erecta were identical. The mutant at the fy locus showed a qualitative difference with Landsberg erecta . Crosses were made between this line and the wild type Landsberg erecta . F2 plants, resulting from autopollination of the hybrid, were analysed and showed no cosegregation between the observed protein and the flowering phenotype, indicating that these two lines differ by more than a single mutation.