磷脂酶Dδ对拟南芥叶片衰老过程中内源ROS和激素含量的影响

活性氧（ROS）和植物激素是植物衰老过程中重要的内在或者外在的调控因子。我们发现,相对于离体诱导的衰老过程,在脱落酸（ABA）和乙烯（ethylene）促进的衰老过程中有较多的活性氧积累;在对拟南芥磷脂酶Dδ（PLDδ）缺失型突变体的研究中发现,与野生型相比,突变体在衰老过程中产生较少的活性氧。我们比较了上述两种基因型的离体叶片在离体、ABA和ethylene三种衰老处理下内源的ABA、茉莉酸甲酯（MeJA）、玉米素核苷（Zeatin Riboside, ZR）和吲哚乙酸（IAA）的含量变化,发现每一种激素对上述三种衰老处理的响应模式都很相似。在离体诱导的衰老中,两种基因型拟南芥的内源激素含量没有差异;而在ABA促进的衰老过程中,PLDδ缺失型突变体叶片中的MeJA的含量较低,ZR和IAA含量较高;在乙烯促进的衰老过程中,突变体中的ABA和MeJA的含量较低,ZR和IAA含量较高。上述内源激素的这种变化可能有助于延缓突变体的衰老。     

分析拟南芥叶片在激素促进的衰老中内源激素变化来解析抑制磷脂酶Dα1延缓激素促进衰老的机制

采后衰老进程在很大程度上受到内源和外源激素的影响。抑制拟南芥中磷脂酶Dα1 (phospholipase Dα1, PLDα1)的表达后,使得外源脱落酸（abscisic acid,ABA）和乙烯加速的离体叶片衰老过程在一定程度上得到了缓解。然而,内源激素在这个过程中的作用尚不清楚。本研究对比分析了野生型和PLDα1缺失型两种基因型拟南芥叶片在3种不同人工老化过程中（离体诱导的、外源ABA和乙烯促进的衰老过程）,内源ABA,茉莉酸甲酯（methyl jasmonate,MeJA）、 吲哚乙酸（indole 3 acetic acid,IAA）、玉米素核苷（zeatin riboside,ZR）和赤霉素（gibberellic acid,GA3）的含量变化。这5种激素对3种不同衰老处理方式的响应模式表明了人工老化过程存在着两个不同阶段,并且在衰老早期每种激素的变化模式相同。PLDα1功能缺失使得激素加速的衰老过程得以延缓,这与内源ABA、MeJA、ZR和IAA的含量变化有关,而与GA3的含量变化无关。同时,ZR和IAA的变化模式也说明了这两种激素的变化可能是缺失PLDα1延缓激素加速的衰老过程这一事件的原因而非结果。     

植物在缺钾胁迫中膜稳定性的变化

植物维持膜的功能是其抵御胁迫的关键问题,而维持膜功能必须要保持膜的稳定性和合适的流动性。我们前期的研究发现植物主要是通过积累叶片膜脂和保持根部膜脂基本不变来适应长期缺钾。在本研究中,以拟南芥和其具有耐受缺钾胁迫特性的近缘种须弥芥为对象,研究了与膜的流动性密切相关的双键指数（double bond index,DBI）的变化,发现长期缺钾条件下,两种植物叶片中总的DBI保持不变,根部总的DBI略有降低。同时研究了与膜稳定性密切相关的溶血磷脂的含量和DGDG/MGDG以及PC/PE这两个比值的变化,发现长期缺钾后拟南芥和须弥芥叶片中溶血磷脂的总量呈上升趋势,根部溶血磷脂总量基本保持不变;无论在对照还是缺钾条件下,拟南芥溶血磷脂的总含量要高于须弥芥。须弥芥叶片具有更高的DGDG/MGDG值,根部具有更高的PC/PE值,说明长期缺钾条件下须弥芥膜的稳定性可能更好。这可能是须弥芥耐缺钾的原因之一。     

水稻离体叶片衰老过程中膜脂组分的变化

水稻离体叶片衰老过程中,膜脂磷脂含量随着叶片离体时间的增加而下降,而质膜透性则随时间的增加而上升。BA,Ni~(2 )能延缓叶片磷脂的丧失,ABA,ACC则加速其含量下降,但它们对磷脂酶D活性影响不大。膜脂脂肪酸组分在叶片衰老过程中也发生着变化,其中亚麻酸(18:3)含量下降,不饱和度降低。ABA,ACC促进亚麻酸含量和不饱和度的下降,BA,Ni~(2 )则有延缓作用。     

磷脂酶Dδ对拟南芥叶片衰老过程中内源ROS和激素含量的影响

活性氧(ROS)和植物激素是植物衰老过程中重要的内在或者外在的调控因子.我们发现,相对于离体诱导的衰老过程,在脱落酸(ABA)和乙烯(ethylene)促进的衰老过程中有较多的活性氧积累;在对拟南芥磷脂酶Dδ (PLDδ)缺失型突变体的研究中发现,与野生型相比,突变体在衰老过程中产生较少的活性氧.我们比较了上述两种基因型的离体叶片在离体、ABA和ethylene三种衰老处理下内源的ABA、茉莉酸甲酯(MeJA)、玉米素核苷(Zeatin Riboside,ZR)和吲哚乙酸(IAA)的含量变化,发现每一种激素对上述三种衰老处理的响应模式都很相似.在离体诱导的衰老中,两种基因型拟南芥的内源激素含量没有差异;而在ABA促进的衰老过程中,PLDδ缺失型突变体叶片中的MeJA的含量较低,ZR和IAA含量较高;在乙烯促进的衰老过程中,突变体中的ABA和MeJA的含量较低,ZR和IAA含量较高.上述内源激素的这种变化可能有助于延缓突变体的衰老.     

大豆种子吸胀冷害抗性与其质体膜脂不饱和度正相关

吸胀冷害是干种子在吸胀阶段遭受低温造成不萌发的现象,结果可能造成农作物损失严重。虽然吸胀过程中细胞膜的修复是关键事件,而且细胞膜在响应水分和温度胁迫中扮演重要角色,但是种子吸胀过程中膜变化的过程,特别是膜流动性变化过程研究较少。本文比较了吸胀冷害耐受型（LX）和敏感型（R5）两个大豆品种在吸胀冷害过程中膜脂不饱和度（double bond index, DBI）的变化,结果发现,LX和R5在常温（25℃）吸胀时变化趋势一致,质体膜脂DBI升高,质体外膜脂中磷脂酰甘油(phosphatidylglycerol, PG)分子DBI下降。LX和R5在低温（4℃）吸胀时DBI变化有很大差异,低温吸胀仅仅延缓了耐受型LX中质体膜脂DBI的升高,但是敏感性R5质体膜脂DBI不仅没有升高反而下降。用浓度33%的聚乙二醇 (polyethylene glycol, PEG)引发没有直接引起DBI变化,但是所引起的细微而显著的变化可能为萌发做好准备。PEG引发处理后的R5在吸胀冷害后第二和第三阶段质体膜脂DBI迅速增加,这个增加模式与LX的DBI增加相似。结果表明,吸胀冷害延缓或者阻滞了质体膜脂不饱和度的升高,大豆种子的吸胀冷害抗性与质体膜脂不饱和度正相关,提高质体膜质DBI可以提高吸胀冷害抗性。     

拟南芥角果衰老过程中膜脂的变化

角果发育对某些物种的生殖发育具有重要的作用。拟南芥种子附着在角果里,角果在早期发育时进行光合作用,角果成熟后开裂散落种子之前,其细胞会经历一个衰老的过程。一般植物细胞在衰老过程中要经历膜脂降解的过程,但是角果细胞衰老过程仍未知。通过比较角果衰老过程中拟南芥野生型(WS)及与膜脂代谢密切相关的磷脂酶Dδ缺失突变体(PLDδ KO)中膜脂分子的组成情况、膜脂含量、相对含量及双键指数值,结果发现,在拟南芥角果衰老过程中：(i)质体膜脂和质体外膜脂显著下降;(ii)不同膜脂降解速率不一样,质体膜脂的降解比质体外膜脂的降解快;(iii)总的双键指数DBI下降;(iv)磷脂酶Dδ缺失突变体(PLDδ KO)的角果膜脂组成的基本水平和变化样式与野生型(WS)非常相似。结果说明,角果在衰老过程中发生了膜脂的激烈降解。据此推测：(i) 膜脂水解产物可能转移到种子中用于储藏脂三酰甘油的合成;(ii) 质体膜脂相对含量下降和质体外膜脂相对含量上升导致了总的DBI下降;(iii) PLDδ参与了角果衰老中的膜脂代谢。     

磷脂酶Dδ缺失加剧UV-B诱导的膜伤害

检测了拟南芥野生型（WS）及磷脂酶Dδ缺失突变体在UV-B辐射下的膜脂分子变化,并比较了二者在紫外辐射下的膜脂含量、双键指数及碳链长度的差异。结果发现,紫外辐射导致植株膜脂发生了降解,其中叶绿体膜脂MGDG和DGDG是膜伤害的主要作用靶点,而且突变体中的膜脂降解比野生型剧烈。上述结果说明磷脂酶Dδ的缺失会加剧紫外辐射诱导的膜伤害,导致植株对紫外辐射更加敏感。     

分析拟南芥叶片在激素促进的衰老中内源激素变化来解析抑制磷脂酶Dα1延缓激素促进衰老的机制

采后衰老进程在很大程度上受到内源和外源激素的影响。抑制拟南芥中磷脂酶Dα1（phospholipaseDtxl,PLDod）的表达后,使得外源脱落酸（abscisic acid,ABA）和乙烯加速的离体叶片衰老过程在一定程度上得到了缓解。然而,内源激素在这个过程中的作用尚不清楚。本研究对比分析了野生型和PLDα1缺失型两种基因型拟南芥叶片在3种不同人工老化过程中（离体诱导的、外源ABA和乙烯促进的衰老过程）,内源ABA,茉莉酸甲酯（methyl jasmonate,MeJA）、吲哚乙酸（indole-3-acetic acid,IAA）、玉米素核苷（zeatin riboside,ZR）和赤霉素（gibberellic acid,GA,）的含量变化。这5种激素对3种不同衰老处理方式的响应模式表明了人工老化过程存在着两个不同阶段,并且在衰老早期每种激素的变化模式相同。PLDα1功能缺失使得激素加速的衰老过程得以延缓,这与内源ABA、MeJA、ZR和IAA的含量变化有关。而与GA、的含量变化无关。同时,ZR和IAA的变化模式也说明了这两种激素的变化可能是缺失PLDα1延缓激素加速的衰老过程这一事件的原因而非结果。     

豌豆叶绿体脂氧合酶活性与叶片衰老及膜脂过氧化作用的关系

豌豆叶绿体脂氧合酶(LOX)活性在连体叶片衰老过程中变化不大。ABA处理离体叶片2d叶绿体LOX活性升高,处理时间延长活性下降。抗氧化剂α-生育酚、谷胱甘肽、没食子酸丙酯抑制豌豆叶绿体LOX活性。脂质过氧化产物丙二醛对豌豆叶绿体LOX和大豆纯LOX-1的活性均有抑制作用,大豆LOX-1能促进离体豌豆叶绿体膜脂过氧化作用。因此,豌豆叶绿体LOX可能参与叶片衰老过程中叶绿体膜结构和功能的改变,又受膜脂过氧化产物的制约。     

Lipid Profiling Demonstrates That Suppressing Arabidopsis Phospholipase Dδ Retards ABA-Promoted Leaf Senescence by Attenuating Lipid Degradation

Senescence is the last phase of the plant life cycle and has an important role in plant development. Degradation of membrane lipids is an essential process during leaf senescence. Several studies have reported fundamental changes in membrane lipids and phospholipase D (PLD) activity as leaves senesce. Suppression of phospholipase Dα1 (PLDα1) retards abscisic acid (ABA)-promoted senescence. However, given the absence of studies that have profiled changes in the compositions of membrane lipid molecules during leaf senescence, there is no direct evidence that PLD affects lipid composition during the process. Here, we show that application of n-butanol, an inhibitor of PLD, and N-Acylethanolamine (NAE) 12∶0, a specific inhibitor of PLDα1, retarded ABA-promoted senescence to different extents. Furthermore, phospholipase Dδ (PLDδ) was induced in leaves treated with ABA, and suppression of PLDδ retarded ABA-promoted senescence in Arabidopsis. Lipid profiling revealed that detachment-induced senescence had different effects on plastidic and extraplastidic lipids. The accelerated degradation of plastidic lipids during ABA-induced senescence in wild-type plants was attenuated in PLDδ-knockout (PLDδ-KO) plants. Dramatic increases in phosphatidic acid (PA) and decreases in phosphatidylcholine (PC) during ABA-induced senescence were also suppressed in PLDδ-KO plants. Our results suggest that PLDδ-mediated hydrolysis of PC to PA plays a positive role in ABA-promoted senescence. The attenuation of PA formation resulting from suppression of PLDδ blocks the degradation of membrane lipids, which retards ABA-promoted senescence.     

Changes of Membrane Stability in Potassium Stressed Plants

The maintenance of membrane function is critical to the ability of plants to resist environmental stresses; specifically, the stability and appropriate fluidity of membranes are crucial to their normal function. We previously demonstrated that plants adapt to long term potassium (K+) deficiency by accumulation of membrane lipids in leaves and maintenance of the lipid composition in roots. In this study, which involved Arabidopsis thaliana and its K+ deficiency tolerant relative Crucihimalaya himalaica, we first calculated the double bond index (DBI) as an indicator of membrane fluidity. After exposure to long term K+ deficiency stress, the DBI of the total lipids in leaves of Athaliana and Chimalaica showed no significant changes, whereas the DBI of the total lipids in the roots of these species showed slight increases. Changes in lysophospholipids (lysoPLs) levels, and digalactosyldiacylglycerol/monogalactosyldiacylglycerol (DGDG/MGDG) and phosphatidylcholine/phosphatidylethanolamine (PC/PE) ratios, all of which strongly reflect membrane stability, were also studied in K+ stressed Athaliana and Chimalaica. After long term K+ deficiency, total lysoPLs levels increased in Athaliana and Chimalaica leaves, but showed no significant changes in roots. DGDG/MGDG and PC/PE ratios were higher in Chimalaica leaves and roots than in those of Athaliana. These results indicate that Chimalaica exhibits superior membrane stability compared with Athaliana. This may explain its superior growth and tolerance under K+ deficient conditions.     

The role of abscisic acid and low temperature in chickpea (Cicer arietinum) cold tolerance. II. Effects on plasma membrane structure and function

The frost hardiness of many plants such as chickpea can be increased by exposure to low non-freezing temperatures and/or the application of abscisic acid (ABA), a process known as frost acclimation. Experiments were conducted to study the response over a 14 d period of enriched plasma membrane fractions isolated from chickpea plants exposed to low temperature and sprayed with exogenous ABA. Measurement of the temperatures inducing 50% foliar cell death (LT50), and subsequent statistical analysis suggest that, like many plants, exposure to low temperatures (5/-2 degrees C; day/night) induces a significant level (P <0.05) of frost acclimation in chickpea when compared with control plants (20/7 degrees C; day/night). Spraying plants with exogenous ABA also increased frost tolerance (P <0.05), but was not as effective as low temperature-induced frost acclimation. Both pre-exposure to low temperatures and pre-treatment with ABA increased the levels of fatty acid desaturation in the plasma membrane (measured as the double bond index, DBI). Exposure of chickpea plants to low temperatures increased the DBI by 15% at day 4 and 19% at day 14 when compared with untreated control plants. Application of ABA alone did not increase the DBI by more than 6% at any time; the effects of both treatments applied together was more than additive, inducing a DBI increase of 27% at day 14 when compared with controls. There was a good correlation (P <0.05) between the DBI and LT50, suggesting that the presence of more unsaturated lipid in the plasma membrane may prevent cell lysis at low temperatures. Both pre-exposure to low, non-freezing temperatures and pre-treatment with ABA induced measurable changes in membrane fluidity, but these changes did not correlate with changes in LT50, suggesting that physical properties of the plasma membrane other than fluidity are involved in frost acclimation in chickpea.     

Profiling the Changes of Molecular Species in Membrane Lipids during Cold acclimation in Winter and Spring Cultivars of Rapeseed (Brassica napus)

The changes of molecular species in membrane lipids during cold acclimation (CA) in winter and spring cultivars of rapeseed (Brassica napus) were profiled with ESI MS/MS based lipidomics. The membrane fluidity, the critical properties for plants to tolerate freeing injuring, was examined with double bond index (DBI) and carbon number of the fatty acid of the glycrolipid. The results indicated that the molecular composition of membrane lipids in the two cultivars under both normal growth condition and CA were similar to that of Arabidopsis reported previously and that DBI in spring cultivar accumulation subtly more than that in winter cultivar. The results suggested that CA of rapeseeds needs more than 3 days and that spring cultivar were more sensitive to CA, which was confirmed by the CA induced proline accumulation.