Methylation changes in specific sequences in response to water deficit

ABSTRACT

Conditions of environmental stress may lead to epigenetic variability in plants. In particular, variation in genome methylation may be a mechanism of plant adaptation to abiotic stresses. We have investigated the relationship between water deficit and DNA changes in specific sequences by using the CRED-RA (Coupled Restriction Enzyme Digestion and Random Amplification) approach. Three polymorphic bands have been detected in dehydrated and rehydrated pea root tips. One of these bands is a zinc-finger sequence with three non canonical motifs, that could be involved in signal transduction pathways.     

Jasmonic Acid is Induced in a Biphasic Manner in Response of Pea Seedlings to Wounding

The role of jasmonic acid (JA) in plant wounding response has been demonstrated. However, the source of JA in wound signaling remains unclear. In the present study, pea seedlings were used as material to investigate the systemic induction of JA and the activation of lipoxygenase (LOX)-dependent octadecanoid pathway upon wounding. The results showed that endogenous JA could induce two peaks in the wounded leaves and the stalks, while only one peak in the systemic leaves.LOX activity and its protein amount were also induced and the stimulation mainly occurred in the late phase, while one peak of induction was present after pretreatment with JA. Applied nordihydroguaiaretic acid (NDGA), an inhibitor of LOX activity, only inhibited the induction of JA in the late phase, and the resistance of pea was impaired. Furthermore, 13(S)-hydroperoxy-9(Z), 11 (E)-octadecadienoic acid (13(S)-H(P)ODE) was confirmed to be the main product of LOX throughout the experimental time. In addition, immunocytochemical analysis also revealed the occurrence of JA biosynthesis and transport upon wounding. These results demonstrated that wound-induced JA in wounded leaves resulted from Its biosynthesis and conversion from its conjugates, while in systemic leaves resulted from its transport and biosynthesis; and proved that the LOX pathway was vital to the wound-induced defense response involved in JA biosynthesis.     

271份豌豆种质资源农艺性状遗传多样性分析

以来自五大洲57个国家和地区的271份豌豆资源为材料,利用主成分分析和聚类分析的方法,评价其2个质量性状和7个数量性状的遗传变异水平。结果表明,参试资源的农艺性状具有丰富的遗传多样性。其中遗传多样性指数最高的是始荚节位(2.0590),其次是主茎节数(2.0421);性状变异系数最大的是小区产量(64.874%),其次是百粒重(61.870%)。采用SPSS 22.0软件对参试资源7个数量性状的主成分分析结果表明,前3个主成分因子累计贡献率达66.021%,Ward法聚类将参试的271份豌豆资源划分为4大类群,其中第Ⅱ类群属于高秆、大粒、高产种质,具有很高的丰产潜力,为我国杂交育种亲本选择提供了原材料。对取材数大于等于5的来自于四大洲17个国家的参试资源进一步分析发现,不同地理区域资源间遗传变异显著,其中印度的遗传多样性指数最高(1.7533),巴基斯坦的最低(0.9685),波兰的变异系数和遗传多样性都较高。综合分析国外种质资源的农艺性状,有助于我国豌豆育种项目亲本选配。     

Jasmonic Acid is Induced in a Biphasic Manner in Response of Pea Seedlings to Wounding

The role of jasmonic acid （JA） in plant wounding response has been demonstrated. However, the source of JA in wound signaling remains unclear. In the present study, pea seedlings were used as material to investigate the systemic induction of JA and the activation of lipoxygenase （LOX）-dependent octadecanoid pathway upon wounding. The results showed that endogenous JA could induce two peaks in the wounded leaves and the stalks, while only one peak in the systemic leaves. LOX activity and its protein amount were also induced and the stimulation mainly occurred in the late phase, while one peak of induction was present after pretreatment with JA. Applied nordihydroguaiaretic acid （NDGA）, an inhibitor of LOX activity, only inhibited the induction of JA in the late phase, and the resistance of pea was impaired. Furthermore, 13（S）- hydroperoxy-9（Z）, 11 （E）-octadecadienoic acid （13（S）-H（P）ODE） was confirmed to be the main product of LOX throughout the experimental time. in addition, immunocytochemical analysis also revealed the occurrence of JA biosynthesis and transport upon wounding. These results demonstrated that wound-induced JA in wounded leaves resulted from its biosynthesis and conversion from its conjugates, while in systemic leaves resulted from its transport and biosynthesis; and proved that the LOX pathway was vital to the wound-induced defense response involved in JA biosynthesis.     

Physiological Responses of Pea Plants to Salinity and Gibberellic Acid

Pea is a seed legume. It is rich in cellulose fibre and protein. It is also a significant source of minerals and vitamins. In this paper, we set out to better characterize the physiological responses of Pisum sativum L. to the combined effects of NaCl, 100 mM and gibberellins (GA3). Our analysis revealed that NaCl caused a decrease in growth resulting in a reduction in root elongation, distribution and density, leaf number and leaf area, and a decrease in dry matter of roots and shoots. However, the contribution of GA3 in the salty environment induced an increase in these different parameters suggesting an improving effect of this hormone on growth of pea in presence of salt. NaCl also led to a disturbance of the photosynthetic machinery. Indeed, level of chlorophyll pigments (a and total) and photosynthetic activity were decreased compared to the control plants. However, the exogenous supply of GA3 restored this decrease in net CO₂ assimilation, but not in chlorophyll content. Additional analyses were performed on the effect of salinity/GA3 interaction on osmolytes (soluble sugars and starch). Our results showed an increase in sugars and a decrease in starch in the presence of 100 mM NaCl. The salt-GA3 combination resulted in compensation of soluble sugar contents but not of starch contents, suggesting a beneficial effect of GA3 under saline stress conditions. Level of three main polyamines putrescine, spermidine, and spermine increased significantly in all organs of salt-treated plants.     

大花蕙兰子房授粉前后基因组DNA 胞嘧啶甲基化状态的MSAP 分析

应用甲基化敏感扩增多态性(Methylation sensitive amplified polymorphism, MSAP) 技术分析了大花蕙兰( Cymbidium hybridium) 授粉前后子房DNA 甲基化状态的变化(甲基化水平和甲基化差异模式) 。采用72 对引物进行选择性扩增, 共得到5892 条带, 其中748 条带为甲基化多态性带。结果显示DNA 甲基化在大花蕙兰子房发育过程中发生频繁, 从授粉前后子房的总扩增位点甲基化水平(14%和11. 4%) 和全甲基化率(9.5%和7.8% ) 来看, 授粉后都略低于未授粉子房, 表明子房在授粉后的发育过程中在某些位点发生了去甲基化。除甲基化水平有变化外, 大花蕙兰子房授粉前后的DNA 甲基化模式也存在较大差异, 共检测到14 种带型, 分为两大类( Ⅰ 和Ⅱ 型)。其中, 授粉前后DNA 甲基化状态保持不变的位点少, 只占25.6% , 归为Ⅰ型; 大部分检测位点( 占74.4% , 归为Ⅱ型) 的DNA 甲基化模式在授粉前后存在显著差异。上述结果表明, 大花蕙兰子房发育过程中以DNA 甲基化为代表的表观遗传调控起重要作用。本研究的开展将促进对与大花蕙兰子房发育相关的甲基化差异片段及受DNA 甲基化调控的关键基因的克隆, 进而为从表观遗传学这一新角度揭示大花蕙兰子房发育的分子机制奠定基础。     

植物DNA甲基化变异对生物和非生物胁迫的响应机制

高等植物具有复杂的机制使其对环境的变化做出响应,这种机制是通过长期进化建立起来的.它们能够对出现的生物和非生物胁迫产生响应.在分子水平上,植物对各种胁迫的响应是受多基因表达变化调控的,包括植物激素水杨酸、脱落酸等信号途径在整合、协调植物胁迫过程中起关键作用.近年来的研究表明,在植物响应胁迫这一过程中还进行着表观遗传调控...     

逆境胁迫对水稻DNA甲基化水平的影响

植物在逆境胁迫下发生复杂的表现遗传变化,包括DNA甲基化、组蛋白修饰和RNA介导的基因沉默等.其中DNA甲基化是表现遗传学中的重要组成部分,主要通过甲基化、去甲基化来参与逆境胁迫下基因表达的调控,进而增强植物体的抗逆性,调节植物体的生长发育.就非生物与生物胁迫对水稻DNA甲基化水平的影响进行综述,为从表观遗传水平研究水稻抗逆性的机制提供理论参考.     

Red-light-induced Changes in the Distribution of Xanthoxin in Pea Seedlings

The distribution of xanthoxin (Xan), was determined in light-grown, 20-d-old pea (Pisum sativum L. cv. Progress No. 9) seedlings. The cis,trans-xanthoxin (c,t-Xan) and the trans,trans-xanthoxin (t,t-Xan) were more abundant in the young leaves and terminal bud; their concentrations in leaves were 2 - 3 times those in internodes of the same nodes. After the onset of red-light-irradiation, the concentration of both Xan isomers in 7-d-old dark-grown pea seedlings increased after a 12-h lag time. The increased level of Xan was greatest in the terminal bud and decreased to lower parts of the seedlings. The ratio of c,t-Xan to t,t-Xan concentration in the seedlings was about 2:3.     

Spatio-temporal Development of Pea Root Rot Disease through Secondary Infections during a Crop Cycle

Spatio‐temporal development of pea root rot was experimentally characterized. Experiments performed under controlled conditions with trap plants located at varying distances from a diseased source plant indicated that disease spread from a diseased plant to a healthy one was generally below 15 cm. A field experiment was conducted to measure disease spread gradients and the rate of secondary infection at two planting densities, 70 and 140 plants/m². Relationships between plant and root disease incidences, and between root disease incidence and root disease severity indicated a strong disease intensification both within and between diseased plants. Measurement of disease gradients indicated that disease did not spread farther than 10 cm from the diseased source plant, i.e. one and two plants away along a row at low and high planting densities respectively. Disease did not spread to neighbouring rows, located 15 cm away. Rates of secondary infection over 14‐day periods ranged between zero (no disease increase) and 0.35 plant/plant/day, and was in general larger in high‐density plots than in low‐density plots. Implications of the results for disease management are discussed.     

Analysis of DNA methylation in different maize tissues

DNA methylation plays an important role in gene expression regulation during biological development and tissue differentiation in plants. This study adopted methylation-sensitive Amplified fragment length polymorphism （AFLP） to compare the levels of DNA cytosine methylation at CCGG sites in tassel, bracteal leaf, and ear leaf from maize inbred lines, 18 White and 18 Red, respectively, and also examined specific methylation patterns of the three tissues. Significant differences in cytosine methylation level among the three tissues and the same changing tendency in two inbred lines were detected. Both MSAP （methylation sensitive amplification polymorphism） ratio and full methylation level were the highest in bracteal leaf, and the lowest in tassel. Meanwhile, different methylation levels were observed in the same tissue from the inbred lines, 18 White and 18 Red. Full methylation of internal cytosine was the dominant type in the maize genome. The differential methylation patterns in the three tissues were observed. In addition, sequencing of nine differentially methylated fragments and the subsequent blast search revealed that the cytosine methylated 5 ＇ -CCGG-3 ＇ sequences were distributed in repeating sequences, in the coding and noncoding regions. Southern hybridization was used to verify the methylation polymorphism. These results clearly demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the maize genome, and the complexity of DNA methylation change during plant growth and development. The different methylation levels may be related to specific gene expression in various tissues.     

Solute Heterogeneity and Osmotic Adjustment in Different Leaf Structures of Semi-Leafless Pea (Pisum sativum L.) Subjected to Water Stress

Abstract: Semi-leafless varieties of pea have considerable agronomic importance and it has been suggested that they may have a superior response to water deficits than conventional varieties. However, these varieties are poorly characterized from a physiological point of view and there is lack of a physiological basis for their supposed better performance under conditions of water deficit. Here, we describe the solute distribution in the different leaf structures of a semi-leafless pea variety ( Pisum sativum L.) under non-limiting water conditions and under water stress. A conventional variety was subjected to the same conditions for comparative purposes. A detailed study was carried out both at the tissue level and at the single cell level. In control conditions, epidermal vacuoles of tendrils showed a different ion distribution of those of the laminar leaf structures. However, under water deficit, only stipules of the semi-leafless variety showed a significantly higher capability to increase osmolarity. This occurred by accumulating potassium, magnesium and chloride to a higher extent than other leaf structures. The inability of performing an adequate osmotic adjustment in tendrils may be the cause of the lack of a better response to water deficit.     

运用MSAP技术测定谷子基因组DNA胞嘧啶甲基化水平

DNA甲基化是真核生物一种重要的表观修饰形式。为了探讨谷子基因组DNA胞嘧啶甲基化的水平和模式,以谷子Setaria italica的两个品种朝谷58号和豫谷1号为实验材料,利用Eco RⅠ和HpaⅡ/MspⅠ双酶切建立适合于谷子基因组的甲基化敏感扩增多态性(MSAP)分析体系。结果表明,从100对MSAP选扩引物中,筛选出32对MSAP引物组合,在朝谷58号和豫谷1号中分别扩增产生1 615、1 482条清晰可辨且可重复的DNA条带,其中包括3种类型的甲基化条带,朝谷58号和豫谷1号的基因组中CCGG序列胞嘧啶甲基化水平分别为6.93%和8.77%。这种谷子不同品种间甲基化水平和分布位点的差异为从表观遗传学的角度培育新品种提供了初步的理论依据和参考。     

Improved PCR-BSP Assay for Multiplex Methylation Pattern Analysis in Minimal Amount of DNA

Cell-specific DNA methylation pattern detection is of great importance for the tumorigenesis and differentiation studies. Comparatively, large amounts of DNA were needed for traditional methods of DNA methylation pattern detection, and therefore, more sensitive method for high throughput analysis with a limited amount of DNA is needed. With Mouse 3T3 cells, we developed new multiplex-nested PCR technologies for bisulfite-assisted genomic sequencing PCR (BSP) methylation pattern detection method. Primers step add-in method and templates precipitation methods efficiently increase the throughput of the assay, and the nested PCR method also increased the sensitivity. The optimized assay could successfully detect 15 sequences of methylation pattern with a minimal amount of DNA (500–1,000 cells of genome DNA).     

DNA甲基化介导的植物逆境应答和胁迫记忆

DNA甲基化是表观遗传修饰的重要形式,它不仅对植物生长发育具有重要的调控作用,而且参与了植物对各种逆境胁迫的应答过程。逆境通过改变植物DNA基化水平和模式对胁迫应答基因网络进行调控,从而增强当代或后代对逆境的适应性。本文主要对DNA甲基化介导的生物和非生物逆境应答及植物胁迫记忆的最新研究进展进行综述,同时对该领域研究中存在的问题和未来研究的方向进行讨论与展望。     

豌豆肌动蛋白异型体的表达与系统发生分析

豌豆(Pisum sativum Linn.)肌动蛋白基因至少可以分为3类异型体: PEAcⅠ、PEAcⅡ和PEAcⅢ,它们的编码区序列相似性很高,而非编码区序列存在显著的差异.RT-PCR和以3′端非翻译区作探针的Southern杂交结果显示豌豆肌动蛋白异型体基因在根、茎、叶、卷须、花粉和幼嫩果实中均表达,但表达强度存在明显差异.利用连续稀释电泳对PEAcⅠ的转录本水平进行分析后发现它倾向于在幼嫩的器官中表达,且在7 d的茎中表达最强;在一个月内的叶片中表达也较强,此后明显下降;卷须中表达变化不显著,在花粉中表达很弱,在幼嫩荚果中的表达亦较多.PEAcⅡ表达特点与PEAcⅠ具有某些相似性.根据肌动蛋白序列重建的系统发生树表明豌豆肌动蛋白3类异型体间的分歧明显,但起源于单、双子叶植物分化前的共同基因祖先.推测豌豆肌动蛋白异型体基因在转录调控和细胞功能上存在差异.     

Water Deficit in Pea Root Tips: Effects on the Cell Cycle and on the Production of Dehydrin-like Proteins

Dehydrin-like proteins have been detected in nuclei and cytoplasmof meristematic root tip cells from pea seedlings subjectedto slow dehydration at 90% relative humidity for 48 h or more.Evidence was gained from Western blotting and immunocytochemicalexperiments using an antibody raised against the conserved domainof dehydrin proteins. Flow cytometer analysis has shown thatcycling cells of root tip meristems from dehydrated seedlingsare mostly arrested in G2 phase. Other stress treatments thoughtto involve water depletion (osmotic stress, cold treatment)or to modulate cell response to water deficit (abscisic acid)gave less clear-cut results with all treatments lowering theproportion of cells entering the S phase, but without a definiteand persistent arrest in any preferential phase of the cycle.Possible interrelationships between G2 arrest and dehydrin productionare discussed. Cell cycle; dehydrins; flow cytometry; nuclei; pea; Pisum sativum L.; water stress     

逆境处理和DNA甲基化影响柑橘体细胞胚发生

对15种柑橘胚性愈伤组织进行体细胞胚诱导,发现逆境处理有利于体细胞胚发生,并可以恢复部分品种的体细胞胚发生能力.对具有和失去体细胞胚发生能力的两种纽荷尔脐橙( Citrus sinensis Osb.)愈伤组织进行随机扩增多态性DNA (RAPD) 分析没有检测到带型的差异,而对它们的甲基化敏感扩增多态性 (MSAP) 进行分析则发现两种愈伤组织间具有明显的DNA甲基化差异,具体细胞胚发生能力的愈伤组织的甲基化水平较失去体细胞胚发生能力的低.