生物钟PRR蛋白促进拟南芥幼苗中花青素的合成

生物钟(circadian clock)是激发植物生理特征节律性表达，并使之维持稳定的保守内源调节机制。PRR(PSEUDO-RESPONSE REGULATOR)蛋白家族是生物钟中央振荡器的重要组成部分，调控植物的种子萌发、下胚轴伸长和开花等多种生命过程。花青素(anthocyanin)是植物次生代谢产物，对植物的繁衍、生长发育和抵抗逆境胁迫具有重要作用。该研究以拟南芥(Arabidopsis thaliana)为对象，探讨生物钟PRR蛋白对花青素生物合成的调控功能和分子机制。结果表明：(1)在PRR基因单突变体及多突变体幼苗中，花青素的积累明显降低，某些花青素合成相关基因的表达也显著降低。(2)相反，在PRR5过表达幼苗中，花青素的积累以及某些花青素合成相关基因的表达则显著升高。(3)蛋白相互作用结果显示，PRR5蛋白能与MYB75、TT8、MYB90及MYB113等花青素调控蛋白相互作用，并形成复合物。(4)遗传学分析结果显示，拟南芥PRR5诱导幼苗中花青素的合成依赖于MYB家族花青素调控蛋白。综上认为，生物钟PRR蛋白可能通过PRR5与MYB75、TT8等相互作用，促进拟南芥幼...     

野生罂粟COR、BBE基因片段融合及其RNAi载体构建

可待因酮还原酶(COR)与小檗碱桥酶(BBE)是吗啡合成代谢途径的关键酶,其活性大小直接影响着吗啡合成途径中生物碱的代谢合成。采用RT-PCR从罂粟幼叶克隆出COR和BBE基因全序列,同源性比较结果显示,它们与GenBank上已报道的COR和BBE基因高度同源。利用blast及分子生物学软件DNAStar对COR和BBE基因的cDNA序列同源性进行分析比较,分别从各基因中筛选和克隆了一段同源性极低、约400～500 bp的片段;并应用重叠PCR法将其拼接成744 bp的融合基因BC,以中间载体pHANNIBAL和植物表达载体pART27为基础,构建了以CaMV 35S启动子驱动的含有“正向BC融合片段- pdk内含子-反向BC融合片段”的ihRNAi植物表达载体,通过转化野生罂粟,初步研究了以COR和BBE基因为靶标的RNAi对内源吗啡合成的抑制效果,为进一步培育低吗啡高蒂巴因的罂粟种质提供了依据。     

Effects of COR6.6 and COR15am polypeptides encoded by COR (cold-regulated) genes of Arabidopsis thaliana on dehydration-induced phase transitions of phospholipid membranes.

Cold acclimation of Arabidopsis thaliana includes the expression of cold-regulated (COR) genes and the accumulation of COR polypeptides. The hydration characteristics of two COR polypeptides, COR6.6 and COR15am, have been determined and their effects on the dehydration-induced liquid crystalline-to-gel and lamellar-to-hexagonal II phase transitions in phospholipid mixtures have been examined. After dehydration at osmotic pressures between 8 and 150 MPa, the water content of the COR polypeptides was less than that of bovine serum albumin, with COr15am the least hydrated: bovine serum albumin > COR6.6 > COR15am. Neither COR6.6 nor COR15am altered the dehydration-induced gel lamellar --> fluid lamellar phase transition temperature of either dipalmitoylphosphatidylcholine or dioleoylphosphatidylcholine (DOPC). In multilamellar vesicles of dioleoylphosphatidylethanolamine:DOPC (1:1, mol:mol) prepared by either freeze-thaw or reverse-phase evaporation methods, neither COR6.6, COR15am, nor bovine serum albumin altered the incidence of the dehydration-induced formation of the inverted hexagonal phase as a function of osmotic pressure. However, a specific ultrastructural alteration--the formation of a striated surface morphology in the lamellar domains--was observed in mixtures of dioleoylphosphatidylethanolamine:DOPC that were dehydrated in the presence of COR15am. Nevertheless, neither COR6.6 nor COR15am appears to participate in a specific protein-phospholipid interaction that alters the dehydration-induced phase behavior of phospholipid vesicles.     

Purification and properties of Arabidopsis thaliana COR (cold-regulated) gene polypeptides COR15am and COR6.6 expressed in Escherichia coli.

Arabidopsis thaliana cold-regulated genes COR15a and COR6.6 encode 15- and 6.6-kD polypeptides, respectively. The COR15a polypeptide is known to be targeted to chloroplasts and, during import, to be processed to a 9.4-kD polypeptide designated COR15am. The COR6.6 polypeptide is thought to be located in the cytosol. The coding sequences for COR15am and COR6.6 were fused to the bacteriophage T7 promoter and expressed in Escherichia coli. The recombinant polypeptides COR15amr and COR6.6r were purified to near homogeneity using a combination of ammonium sulfate fractionation, ion-exchange chromatography, and adsorption chromatography on hydroxyapatite. COR15amr and the major species of COR15am in chloroplasts co-migrated on both two-dimensional O'Farrell gels and nondenaturing polyacrylamide gels. These data corroborate the site of COR15a processing and indicate no difference in charge or quaternary structure between COR15amr and the major species of COR15am in plants. In contrast, the migration patterns of COR6.6r and COR6.6 on two-dimensional gels suggest that a considerable portion of the COR6.6 population in plants is modified. In the accompanying papers (M.S. Webb, S.J. Gilmour, M.F. Thomashow, P.L. Steponkus [1996] Plant Physiology 111: 301-312; M. Uemura, S.J. Gilmour, M.F. Thomashow, P.L. Steponkus [1996] Plant Physiology 111: 313-327), the effects of COR15amr and COR6.6r on the cryostability and lyotropic phase behavior of liposomes are examined.     

葡萄COR27基因的克隆与抗寒功能研究

冬季低温是制约我国葡萄和葡萄酒产业发展的主要因素之一,揭示葡萄在冷胁迫下的信号转导通路、挖掘抗寒相关基因并解析其功能,对高耐寒品种的培育具有重要的理论和应用价值。本研究在欧亚种‘玫瑰香’葡萄(Vitis vinifera L.‘Muscat Hamburg’)冷胁迫相关转录组分析的基础上,鉴定了一个抗寒候选基因,通过同源性分析将其命名为VvCOR27。VvCOR27基因的cDNA序列(1082 bp)中,其开放阅读框(ORF)为909 bp,编码302个氨基酸。同源性分析显示,13个物种的COR27蛋白均具有3个特有的保守结构域。定量RT-PCR分析表明,VvCOR27在4℃低温处理24 h后大量表达。基于基因组序列的启动子基序分析表明,VvCOR27启动子区均只含有1个EE、EEL、G-box、ABREL元件,其数量少于AtCOR27,这可能是VvCOR27响应冷胁迫较AtCOR27滞后的原因。对3个超表达VvCOR27转基因拟南芥株系的抗寒性鉴定表明,VvCOR27参与了植株对冷胁迫的应答并作为正调控因子增强了植株对冷胁迫的耐受能力。     

Expression analysis and functional characterization of a cold-responsive gene COR15A from Arabidopsis thaliana

The cold-responsive (COR) genes play an important role in cold acclimation of higher plants. Here, a tight correlation between chloroplast functionality and COR15A expression, and the functional characterization of Arabidopsis COR15A involved in salt/osmotic stress, were revealed. COR15A gene is light inducible and expressed in light-grown seedlings. The expression level of COR15A was reduced when chloroplasts were damaged by norflurazon treatment. By using several albino mutants, seca1, secy1, and tic20, all of which exhibited severe defects in both structure and function of chloroplast, it was shown that the accumulation of COR15A mRNA depends on chloroplast functionality. Real-time RT-PCR and GUS-staining assays demonstrated that COR15A was induced by salt/osmotic stress partially via ABA. Overexpression of COR15A in Arabidopsis resulted in the seedlings displaying hypersensitivity to salt/osmotic stress. All these results suggest that plant acquire the ability to fully express COR15A only after the development of functional chloroplasts, COR15A may be involved in response to salt/osmotic stress during early stages of plant development.     

Characterization of circadian-associated pseudo-response regulators: II. The function of PRR5 and its molecular dissection in Arabidopsis thaliana

Together with PRR1/TOC1, PRR5 belongs to the small family of Pseudo-Response Regulators (PRRs), which function as clock components of Arabidopsis thaliana. We employed a set of transgenic lines, each of which was designed to misexpress a truncated form of the PRR5 molecule, together with the original transgenic line (named PRR5-ox) that misexpresses the entire PRR5 polypeptide. The results of genetic analysis suggested that PRR5-ox seedlings showed a phenotype of hypersensitivity to red light during early photomorphogenesis in a manner dependent on red light photoreceptors (PhyA and PhyB), but independent of PRR1/TOC1. The set of newly constructed transgenic lines (named PRR5-N-ox and PRR5-C-ox) were also characterized in terms of circadian-associated phenotypes. The results suggest that the N-terminal pseudo-receiver domain of the PRR5 molecule seems to be dispensable for the misexpressed PRR5 molecule to bring about the phenotype of red light sensitivity. However, PRR5-N-ox plants, misexpressing only the pseudo-receiver domain, showed a phenotype of long period of free-running circadian rhythms of certain clock-controlled genes. Considering these and other results, we discuss the structure and function of PRR5 in the context of current views of the circadian clock in higher plants.     

Characterization of Circadian-associated pseudo-response regulators: I. Comparative studies on a series of transgenic lines misexpressing five distinctive PRR Genes in Arabidopsis thaliana

Every member of a small family of Pseudo-Response Regulator (PRR) genes, including Timing of Cab Expression 1 (TOC1 [or PRR1]), are believed to play roles close to the circadian clock in the model higher plant Arabidopsis thaliana. In this study we established a transgenic line that misexpresses (or overexpresses) the PRR7 gene. As compared with wild-type plants, the resulting PRR7-misexpressing plants (designated PRR7-ox) showed characteristic phenotypes as to hallmarked circadian-associated biological events: (i) early flowering in a manner independent of photoperiodicity, (ii) hypersensitive response to red light during early photomorphogenesis, and (iii) altered free-running rhythms with long period of clock-associated genes. Finally, a series of all transgenic lines (PRR1-ox, PRR3-ox, PRR5-ox, PRR7-ox, and PRR9-ox) were characterized comparatively with regard to their clock-associated roles. The results suggested that the five homologous PRR factors play coordinate roles, distinctively from one another, and closely to the circadian clock in higher plants.     

LIGHT-REGULATED WD1 and PSEUDO-RESPONSE REGULATOR9 form a positive feedback regulatory loop in the Arabidopsis circadian clock

In Arabidopsis thaliana, central circadian clock genes constitute several feedback loops. These interlocking loops generate an ~24-h oscillation that enables plants to anticipate the daily diurnal environment. The identification of additional clock proteins can help dissect the complex nature of the circadian clock. Previously, LIGHT-REGULATED WD1 (LWD1) and LWD2 were identified as two clock proteins regulating circadian period length and photoperiodic flowering. Here, we systematically studied the function of LWD1/2 in the Arabidopsis circadian clock. Analysis of the lwd1 lwd2 double mutant revealed that LWD1/2 plays dual functions in the light input pathway and the regulation of the central oscillator. Promoter:luciferase fusion studies showed that activities of LWD1/2 promoters are rhythmic and depend on functional PSEUDO-RESPONSE REGULATOR9 (PRR9) and PRR7. LWD1/2 is also needed for the expression of PRR9, PRR7, and PRR5. LWD1 is preferentially localized within the nucleus and associates with promoters of PRR9, PRR5, and TOC1 in vivo. Our results support the existence of a positive feedback loop within the Arabidopsis circadian clock. Further mechanistic studies of this positive feedback loop and its regulatory effects on the other clock components will further elucidate the complex nature of the Arabidopsis circadian clock.     

Interaction of two intrinsically disordered plant stress proteins (COR15A and COR15B) with lipid membranes in the dry state

COR15A and COR15B form a tandem repeat of highly homologous genes in Arabidopsis thaliana. Both genes are highly cold induced and the encoded proteins belong to the Pfam LEA_4 group (group 3) of the late embryogenesis abundant (LEA) proteins. Both proteins were predicted to be intrinsically disordered in solution. Only COR15A has previously been characterized and it was shown to be localized in the soluble stroma fraction of chloroplasts. Ectopic expression of COR15A in Arabidopsis resulted in increased freezing tolerance of both chloroplasts after freezing and thawing of intact leaves and of isolated protoplasts frozen and thawed in vitro. In the present study we have generated recombinant mature COR15A and COR15B for a comparative study of their structure and possible function as membrane protectants. CD spectroscopy showed that both proteins are predominantly unstructured in solution and mainly α-helical after drying. Both proteins showed similar effects on the thermotropic phase behavior of dry liposomes. A decrease in the gel to liquid-crystalline phase transition temperature depended on both the unsaturation of the fatty acyl chains and lipid headgroup structure. FTIR spectroscopy indicated no strong interactions between the proteins and the lipid phosphate and carbonyl groups, but significant interactions with the galactose headgroup of the chloroplast lipid monogalactosyldiacylglycerol. These findings were rationalized by modeling the secondary structure of COR15A and COR15B. Helical wheel projection indicated the presence of amphipathic α-helices in both proteins. The helices lacked a clear separation of positive and negative charges on the hydrophilic face, but contained several hydroxylated amino acids.     

Gene regulation and signal transduction in the ICE–CBF–COR signaling pathway during cold stress in plants

Low temperature is an abiotic stress that adversely affects the growth and production of plants. Resistance and adaptation of plants to cold stress is dependent upon the activation of molecular networks and pathways involved in signal transduction and the regulation of cold-stress related genes. Because it has numerous and complex genes, regulation factors, and pathways, research on the ICE–CBF–COR signaling pathway is the most studied and detailed, which is thought to be rather important for cold resistance of plants. In this review, we focus on the function of each member, interrelation among members, and the influence of manipulators and repressors in the ICE–CBF–COR pathway. In addition, regulation and signal transduction concerning plant hormones, circadian clock, and light are discussed. The studies presented provide a detailed picture of the ICE–CBF–COR pathway.