水稻ES1参与生物钟基因表达调控以及逆境胁迫响应

生物钟参与调控植物所有的生长阶段和发育活动。维持植物生物钟稳定的基因在这一过程中起着决定性作用。在克隆了ES1 (EARLY SENESCENCE 1)基因并证明该基因影响水稻(Oryza sativa)叶片失水的基础上, 以前期分离得到的水稻突变体es1-1作为研究对象, 对es1-1及其野生型(日本晴)苗期的地上部分和地下部分进行基因芯片分析。结果表明, es1-1主要的上调基因有42个, 下调基因有14个, 这些差异基因涉及24种代谢途径, 包括调节水稻生物钟的途径(4个)、甲烷代谢途径(3个)和苯基丙氨酸代谢途径(3个)等。进一步对水稻生物钟相关基因进行表达图谱分析, 结果表明, 与野生型相比, es1-1中生物钟相关基因出现了不同程度的差异表达。对es1-1和野生型进行冷胁迫处理, 结果表明es1-1表现更加耐冷, 且冷处理后生物钟基因在日本晴(NPB)和es1-1中都表现出不同程度的差异表达。此外, 在分蘖盛期接种白叶枯菌, 发现es1-1对特定的白叶枯菌具有一定的抗性。由此推测ES1基因参与调控水稻生物钟基因的表达以及响应水稻部分逆境胁迫, 这为更深入研究水稻生物钟基因提供了新线索。     

Bioinformatic and expression analyses on carotenoid dioxygenase genes in fruit development and abiotic stress responses in <Emphasis Type="Italic">Fragaria vesca</Emphasis>

Carotenoid dioxygenases, including 9-cis-epoxycarotenoid dioxygenases (NCEDs) and carotenoid cleavage dioxygenases (CCDs), can selectively cleave carotenoids into various apocarotenoid products that play important roles in fleshy fruit development and abiotic stress response. In this study, we identified 12 carotenoid dioxygenase genes in diploid strawberry Fragaria vesca, and explored their evolution with orthologous genes from nine other species. Phylogenetic analyses suggested that the NCED and CCDL groups moderately expanded during their evolution, whereas gene numbers of the CCD1, CCD4, CCD7, and CCD8 groups maintained conserved. We characterized the expression profiles of FveNCED and FveCCD genes during flower and fruit development, and in response to several abiotic stresses. FveNCED1 expression positively responded to osmotic, cold, and heat stresses, whereas FveNCED2 was only induced under cold stress. In contrast, FveNCED2 was the unique gene highly and continuously increasing in receptacle during fruit ripening, which co-occurred with the increase in endogenous abscisic acid (ABA) content previously reported in octoploid strawberry. The differential expression patterns suggested that FveNCED1 and FveNCED2 were key genes for ABA biosynthesis in abiotic stress responses and fruit ripening, respectively. FveCCD1 exhibited the highest expression in most stages of flower and fruit development, while the other FveCCDs were expressed in a subset of stages and tissues. Our study suggests distinct functions of FveNCED and FveCCD genes in fruit development and stress responses and lays a foundation for future study to understand the roles of these genes and their metabolites, including ABA and other apocarotenoid products, in the growth and development of strawberry.     

Diurnal Oscillations of Soybean Circadian Clock and Drought Responsive Genes

Rhythms produced by the endogenous circadian clock play a critical role in allowing plants to respond and adapt to the environment. While there is a well-established regulatory link between the circadian clock and responses to abiotic stress in model plants, little is known of the circadian system in crop species like soybean. This study examines how drought impacts diurnal oscillation of both drought responsive and circadian clock genes in soybean. Drought stress induced marked changes in gene expression of several circadian clock-like components, such as LCL1-, GmELF4- and PRR-like genes, which had reduced expression in stressed plants. The same conditions produced a phase advance of expression for the GmTOC1-like, GmLUX-like and GmPRR7-like genes. Similarly, the rhythmic expression pattern of the soybean drought-responsive genes DREB-, bZIP-, GOLS-, RAB18- and Remorin-like changed significantly after plant exposure to drought. In silico analysis of promoter regions of these genes revealed the presence of cis-elements associated both with stress and circadian clock regulation. Furthermore, some soybean genes with upstream ABRE elements were responsive to abscisic acid treatment. Our results indicate that some connection between the drought response and the circadian clock may exist in soybean since (i) drought stress affects gene expression of circadian clock components and (ii) several stress responsive genes display diurnal oscillation in soybeans.     

Does the circadian system regulate lactation?

Environmental variables such as photoperiod, heat, stress, nutrition and other external factors have profound effects on quality and quantity of a dairy cow's milk. The way in which the environment interacts with genotype to impact milk production is unknown; however, evidence from our laboratory suggests that circadian clocks play a role. Daily and seasonal endocrine rhythms are coordinated in mammals by the master circadian clock in the hypothalamus. Peripheral clocks are distributed in every organ and coordinated by signals from the master clock. We and others have shown that there is a circadian clock in the mammary gland. Approximately 7% of the genes expressed during lactation had circadian patterns including core clock and metabolic genes. Amplitude changes occurred in the core mammary clock genes during the transition from pregnancy to lactation and were coordinated with changes in molecular clocks among multiple tissues. In vitro studies using a bovine mammary cell line showed that external stimulation synchronized mammary clocks, and expression of the core clock gene, BMAL1, was induced by lactogens. Female clock/clock mutant mice, which have disrupted circadian rhythms, have impaired mammary development and their offspring failed to thrive suggesting that the dam's milk production was not adequate enough to nourish their young. We envision that, in mammals, during the transition from pregnancy to lactation the master clock is modified by environmental and physiological cues that it receives, including photoperiod length. In turn, the master clock coordinates changes in endocrine milieu that signals peripheral tissues. In dairy cows, it is clear that changes in photoperiod during the dry period and/or during lactation influences milk production. We believe that the photoperiod effect on milk production is mediated, in part by the 'setting' of the master clock with light, which modifies peripheral circadian clocks including the mammary core clock and subsequently impacts milk yield and may impact milk composition.     

Entrainment of eclosion and preliminary ontogeny of circadian clock gene expression in the flesh fly,Sarcophaga crassipalpis

Timing of circadian activities is controlled by rhythmic expression of clock genes in pacemaker neurons in the insect brain. Circadian behavior and clock gene expression can entrain to both thermoperiod and photoperiod but the availability of such cues, the organization of the brain, and the need for circadian behavior change dramatically during the course of insect metamorphosis. We asked whether photoperiod or thermoperiod entrains the clock during pupal and pharate adult stages by exposing flies to different combinations of thermoperiod and photoperiod and observing the effect on the timing of adult eclosion. This study used qRT-PCR to examine how entrainment and expression of circadian clock genes change during the course of development in the flesh fly, Sarcophaga crassipalpis. Thermoperiod entrains expression of period and controls the timing of adult eclosion, suggesting that the clock gene period may be upstream of the eclosion pathway. Rhythmic clock gene expression is evident in larvae, appears to cease during the early pharate adult stage, and resumes again by the time of adult eclosion. Our results indicate that both patterns of clock gene expression and the cues to which the clock entrains are dynamic and respond to different environmental signals at different developmental stages in S. crassipalpis.     

Osmotic stress at the barley root affects expression of circadian clock genes in the shoot

The circadian clock is an important timing system that controls physiological responses to abiotic stresses in plants. However, there is little information on the effects of the clock on stress adaptation in important crops, like barley. In addition, we do not know how osmotic stress perceived at the roots affect the shoot circadian clock. Barley genotypes, carrying natural variation at the photoperiod response and clock genes Ppd‐H1 and HvELF3, were grown under control and osmotic stress conditions to record changes in the diurnal expression of clock and stress‐response genes and in physiological traits. Variation at HvELF3 affected the expression phase and shape of clock and stress‐response genes, while variation at Ppd‐H1 only affected the expression levels of stress genes. Osmotic stress up‐regulated expression of clock and stress‐response genes and advanced their expression peaks. Clock genes controlled the expression of stress‐response genes, but had minor effects on gas exchange and leaf transpiration. This study demonstrated that osmotic stress at the barley root altered clock gene expression in the shoot and acted as a spatial input signal into the clock. Unlike in Arabidopsis, barley primary assimilation was less controlled by the clock and more responsive to environmental perturbations, such as osmotic stress.     

Identification of valid reference genes for circadian gene-expression studies in human mammary epithelial cells

ABSTRACT

The circadian clock controls most of the physiological processes in the body throughout days and nights’ alternation. Its dysregulation has a negative impact on many aspects of human health, such as obesity, lipid disorders, diabetes, skin regeneration, hematopoiesis and cancer. To date, poor is known on the molecular mechanisms that links mammary gland homeostasis to the circadian clock but recent reports highlight the importance of loss of circadian genes for mammary gland development and during tumour progression in breast cancer. Gene expression studies are then required to clarify how the circadian clock can modulates the human mammary gland development during ontology and its behaviour in physiological and oncogenic context. For this, in addition to genome-wide studies, real-time quantitative RT-PCR (qPCR) is a powerful and pertinent technique to quantify the expression of a reduced set of genes of interest in many different samples. Relative quantification of qPCR data requires the use of reference genes for normalisation. For circadian studies, reference genes expression must not oscillate in mirror of the circadian clock and must not be affected by the synchronisation protocols required in vitro to reset the circadian clock. Inappropriate selection of reference genes can consequently affect the amplitude of gene expression oscillation and bias data interpretation. Currently, no standard reference genes have been validated regarding these criteria for human mammary epithelial cells and the purpose of this study was to fill this gap. For this, we used the RefFinder tool, which combines four different algorithms, on 9 candidate reference genes. We compared reference genes stability using three different synchronisation protocols applied on four different mammary epithelial cell lines. This allowed us to define a set of reference genes in human mammary epithelial cells whose expression remains stable despite synchronisation protocols. We observed that the synchronisation of cells by serum shock was the most suitable procedure for maintaining the amplitude of oscillation of clock genes over time and we identified RPL4, RPLP0, HSPCB and TBP as an optimal combination of reference genes for the normalisation of the oscillatory expression of clock genes in human mammary epithelial cells.     

Genome-wide transcriptional orchestration of circadian rhythms in Drosophila

Circadian rhythms govern the behavior, physiology, and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and in mammals. To study how gene expression is globally regulated by the clock mechanism, we used a high density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions, and among these the expression of 115 genes was still cycling in constant darkness, i.e. under free-running conditions. Unexpectedly the expression of a large number of genes cycled exclusively under constant darkness. We found that cycling in most of these genes was lost in the arrhythmic Clock (Clk) mutant under light-dark conditions. Expression of periodically regulated genes is coordinated locally on chromosomes where small clusters of genes are regulated jointly. Our findings reveal that many genes involved in diverse functions are under circadian control and reveal the complexity of circadian gene expression in Drosophila.     

Clock is important for food and circadian regulation of macronutrient absorption in mice

Clock genes respond to external stimuli and exhibit circadian rhythms. This study investigated the expression of clock genes in the small intestine and their contribution in the regulation of nutrient absorption by enterocytes. We examined expression of clock genes and macronutrient transport proteins in the small intestines of wild-type and Clock mutant (Clk^mt/mt) mice with free or limited access to food. In addition, we studied absorption of macronutrients in these mice. Intestinal clock genes show circadian expression and respond to food entrainment in wild-type mice. Dominant negative Clock in Clk^mt/mt mice disrupts circadian expression and food entrainment of clock genes. The absorption of lipids and monosaccharides was high in Clk^mt/mt mice whereas peptide absorption was reduced. Molecular studies revealed that Clock regulates several transport proteins involved in nutrient absorption. Clock plays an important role in light and food entrainment of intestinal functions by regulating nutrient transport proteins. Disruptions in intestinal circadian activity may contribute to hyperlipidemia and hyperglycemia.     

干旱对水稻生物钟基因和干旱胁迫响应基因每日节律性变化的影响

内源生物钟的节律运动不仅调控植物的生长发育,而且在调控植物响应和适应环境过程中发挥重要的作用。为了解水稻(Oryza sativa L.)干旱胁迫响应基因和生物钟基因在干旱条件下每日表达变化情况,本文利用实时荧光定量PCR方法研究旱稻品种IRAT109在干旱胁迫下相关基因的表达变化。结果表明,干旱胁迫导致早晨生物钟基因OsPRRs、OsLHY和OsZTL1的表达量显著下降,振幅减弱;同时导致夜晚生物钟基因OsTOC1、OsGI和OsELF3整体表达量升高,振幅增强,但对OsFKF1基因影响不大。同样,大部分水稻干旱胁迫响应基因在干旱胁迫后整体表达量显著升高,但OsDST基因表达量下降;同时大部分抗逆基因周期性表达被扰乱,但OsCIPK12、OsCDPK7和OsDREB1A依然保持24 h内震荡。本研究结果表明干旱胁迫能影响生物钟元件的基因表达,这种互相影响改变了部分基因每日的震荡变化。