磷脂酶Dβ在拟南芥低温信号中的转导作用

磷脂酶D(PLD)不仅是植物中一类主要的磷脂水解酶,而且是一类重要的跨膜信号转导酶类.PLD的磷脂降解功能和信号转导功能均影响植物的抗冻性.本研究以PLDβ基因被敲除的拟南芥突变体及其野生型植株为材料,进行低温驯化和冻害胁迫处理,并分析其作用途径.结果表明,PLDβ基因介导低温信号转导作用,参与渗透调节途径中脯氨酸的调控和抗氧化系统中过氧化氢酶(CAT)活性的调控,并且与低温信号激素ABA不在同一条信号转导途径.本研究为探索通过调控PLD的活性提高植物抗冻性提供了新的途径,并为深入揭示植物的抗冻机理以及磷脂信号转导机制提供实验支持.     

低温胁迫下脱落酸对线粒体膜磷脂酶D活性的影响

高山离子芥(Choraspora bungeana)是一种稀有高山冰缘植物,其生活环境具有低温、强紫外线等胁迫因子。PLD在膜磷脂降解及磷脂信号转导过程中发挥着重要作用,但其活性往往受到多种因素的影响。该研究以高山离子芥试管苗为材料,研究了4℃、0℃和-4℃胁迫下,ABA对高山离子芥试管苗叶中线粒体膜结合态PLD活性的影响。结果表明:10,50和100μmol·L~(-1)脱落酸(ABA)处理高山离子芥后,线粒体膜结合态PLD活性均较未添加ABA的处理组线粒体膜结合态PLD活性高,其中以50μmol·L~(-1) ABA对离子芥叶中线粒体膜结合态PLD活性的促进作用最为显著;外施0.3 mmol·L~(-1)的ABA合成抑制剂钨酸钠处理高山离子芥后,线粒体膜结合态PLD活性较对照组线粒体膜结合态PLD活性降低;在50μmol·L~(-1) ABA+5 mmol·L~(-1) EGTA处理组中,高山离子芥叶中线粒体膜结合态PLD活性低于未添加EGTA处理组线粒体膜结合态PLD活性;在0.3 mmol·L~(-1)钨酸钠+10 mmol·L~(-1)CaCl_2处理组中,高山离子芥叶中线粒体膜结合态PLD活性高于未添加CaCl_2处理组线粒体膜结合态PLD活性。由此推测,低温胁迫下ABA可能通过Ca~(2+)介导影响高山离子芥叶中线粒体膜结合态PLD的活性。     

磷脂酶D信号转导与植物耐盐研究进展

土壤盐害是一个全球性的生态问题,对生态环境和农业生产带来了巨大的负面影响。研究发现,植物磷脂酶D(Phospholipase D,PLD)是磷脂代谢和应答非生物胁迫的重要酶类;PLD具有不同的结构、生化和调节特性,产生信号分子磷脂酸(Phosphatidic acid,PA)并参与多种胁迫反应。总结了PLD及其产物PA调控植物耐盐的相关报道,探讨其感受、应答盐信号的分子机制,为研究植物应答高盐胁迫和农作物分子遗传改良提供相关参考。     

磷脂酶D的细胞信号转导作用

磷脂酶D(PLD)是一类重要的跨膜信号转导酶类.分别由一个基因家族的不同成员编码.植物PLD的总体域结构相似,只是不同类型之间在某些单元上有重要差异.它们各具独特的生物化学特性.不同的PLD在不同的胁迫类型启动的特定的细胞过程中执行独特的细胞信号转导功能.PLD与其它磷脂酶及Ca2 信使之间有交互作用,形成复杂的信号转导网络.这一网络在不同植物种类、器官、组织和细胞类型中表现出特异性.文章最后讨论了PLD研究中有待揭示的问题并展望了今后的发展方向.     

儿茶素诱导的拟南芥根细胞膜脂变化

儿茶素是一种可以短时间内杀死植物细胞的植物毒素,由于具有强的植物毒性,儿茶素是开发除草剂的理想化合物,它可以诱导植物根系统的死亡.为了研究植物根细胞膜脂对化学胁迫的响应规律,我们运用高通量的脂类组学方法检测了拟南芥根中膜脂分子的组成,比较了儿茶素处理下拟南芥野生型(WS)及磷脂酶Dδ缺失突变体( PLDδ-KO)根中膜脂分子的组成情况、膜脂含量、双键指数及碳链长度值.结果发现,儿茶素处理拟南芥根90 min后,二半乳糖基二酰甘油(DGDG)、单半乳糖基二酰甘油(MGDG)、磷脂酰甘油(PG)、磷脂酰胆碱(PC)及磷脂酰肌醇(PI)的总含量在WS与PLDδ-KO植株根中都显著下降,磷脂酰乙醇胺(PE)和磷脂酰丝氨酸(PS)在WS中下降,在PLDδ-KO中上升.儿茶素处理导致PLDδ-KO植株的PC/PE比值显著下降,WS植株PS碳链长度显著增加.上述结果说明儿茶素处理后,磷脂酶Dδ缺失突变体膜不稳定性增加,PLDδ-KO植株对儿茶素胁迫更加敏感.     

拟南芥中磷脂酶Dγ2的低温信号转导作用途径分析

磷脂酶D(PLD)的磷脂降解功能和信号转导功能均能影响植物的抗冻性.本研究以PLDγ2基因被敲除的拟南芥突变体及其野生型为材料,进行低温驯化和冻害胁迫处理,随后由这两种植株的表型及其离子渗透率等来分析PLDγ2基因对拟南芥抗冻性.结果发现,经直接冻害处理后,PLDγ2敲除型的抗冻性与野生型基本一致;但经过低温驯化后的冻害处理,PLDγ2敲除型的抗冻性弱于野生型,即表明PLDγ2基因参与了低温信号转导作用.进一步的实验结果表明PLDγ2基因介导脯氨酸调控的可溶性物质调控途径和过氧化物酶(POD)活性调控的抗氧化系统途径;但是与低温信号激素ABA不在同一条信号转导途径.     

磷脂酶D（PLD）基因的结构、表达及其表达产物在信号转导中的作用

磷脂酶D(PLD EC 3.1.4.4)水解磷脂(PL),磷脂构成生物膜的骨架, 磷脂酶的激活不仅对细胞的结构和稳定性有很重要的作用,而且调控许多重要的细胞生理功能,例如PLD在信号转导、小泡运输、有丝分裂 、激素作用的发挥、细胞骨架组装、防御反应以及种子萌发和衰老过程中都起重要作用。近年来它在跨膜信号转导中的重要作用，越来越引起人们的重视，成为新的研究热点。介绍了磷脂酶基因的结构特点、亚细胞定位、表达的激活抑制以及其表达产物作为胞内信号分子在植物信号转导中的重要作用。     

枇杷幼果PLD和LOX对低温胁迫的响应

以3年生枇杷品种‘早钟6号’(Eriobotrya japonica‘Zaozhong No.6’)容器嫁接苗为试材,于0℃、-1℃、-3℃人工气候室内进行低温胁迫处理,探讨枇杷幼果细胞膜磷脂及相关酶对低温胁迫的响应机制。结果显示,在不同温度胁迫过程中,枇杷幼果磷脂酶D(PLD,EC 3.1.4.4)和脂氧合酶(LOX,EC 1.13.11.12)活性均呈上升趋势;质膜磷脂酰胆碱(PC)和磷脂酰肌醇(PI)含量因逐渐被降解而呈下降趋势,磷脂酸(PA)含量出现积累、增加,而膜结合Ca2+含量有不同程度的降低。随处理时间的延长和处理温度的降低,枇杷幼果细胞PLD和LOX活性增幅加大,从而加速了膜PC和PI的降解和PA的积累。低温胁迫过程中幼果细胞膜PC含量的降幅大于PI,膜结合Ca2+含量的变化与PLD和LOX活性变化呈负相关。低温胁迫下枇杷幼果细胞膜结合Ca2+含量的减少诱导了膜脂降解酶PLD和LOX活性的提高,并导致膜结构稳定性下降,加剧了低温胁迫对膜脂的降解和脂质过氧化伤害,其中尤以-3℃胁迫处理4~6 h对幼果细胞质膜的伤害最严重。表明低温胁迫下Ca2+·Ca M信使系统可能参与枇杷幼果细胞膜PLD和LOX活性的调控。     

李斯特菌侵染宿主细胞过程中磷脂酶D活性变化的初步研究

磷脂酶D(phospholipase,PLD)在感染和炎症反应中发挥重要作用,然而其在单核增生李斯特菌(简称李斯特菌)侵染非洲绿猴肾细胞(Vero)中是否被激活尚未见报道。对李斯特菌刺激下的Vero细胞内PLD的活性变化进行了初步研究。以李斯特菌、佛波醇PMA分别刺激正常细胞及高效表达PLD2-K758R功能缺陷蛋白腺病毒预感染的Vero细胞,研究Vero细胞内PLD活性变化。结果发现,与对照组相比(不给予刺激),在李斯特菌和佛波醇PMA刺激正常Vero细胞过程中,胞内PLD活性增强非常显著。mPLD2-K758R蛋白的过度表达对Vero细胞的PLD基础活性无影响,但对李斯特菌和PMA诱导的PLD激活有明显抑制作用。这初步表明,在李斯特菌诱导的Vero细胞对其吞噬过程中的确伴有PLD的激活,并且可能主要是PLD2亚型被激活,但此吞噬过程中PLD的激活机制及激活后PLD的具体功能尚有待进一步研究。     

植物磷脂酶D基因表达与衰老的关系

磷脂酶D (PLD)是一种重要的磷脂水解酶,在植物细胞中普遍存在。磷脂酶D能激活许多重要的细胞生理功能,包括调控细胞膜的重建、跨膜信号传导及细胞内调控、细胞骨架组装、防御反应以及种子萌发和植物的衰老等。对磷脂酶D的基本特性、磷脂酶D基因特异性表达模式及其活性抑制与植物衰老的关系进行了综述,并探讨和展望了今后植物磷脂酶D基因的研究方向。     

Antisense suppression of phospholipase D alpha retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves.

Membrane disruption has been proposed to be a key event in plant senescence, and phospholipase D (PLD; EC 3.1.4.4) has been thought to play an important role in membrane deterioration. We recently cloned and biochemically characterized three different PLDs from Arabidopsis. In this study, we investigated the role of the most prevalent phospholipid-hydrolyzing enzyme, PLD alpha, in membrane degradation and senescence in Arabidopsis. The expression of PLD alpha was suppressed by introducing a PLD alpha antisense cDNA fragment into Arabidopsis. When incubated with abscisic acid and ethylene, leaves detached from the PLD alpha-deficient transgenic plants showed a slower rate of senescence than did those from wild-type and transgenic control plants. The retardation of senescence was demonstrated by delayed leaf yellowing, lower ion leakage, greater photosynthetic activity, and higher content of chlorophyll and phospholipids in the PLD alpha antisense leaves than in those of the wild type. Treatment of detached leaves with abscisic acid and ethylene stimulated PLD alpha expression, as indicated by increases in PLD alpha mRNA, protein, and activity. In the absence of abscisic acid and ethylene, however, detached leaves from the PLD alpha-deficient and wild-type plants showed a similar rate of senescence. In addition, the suppression of PLD alpha did not alter natural plant growth and development. These data suggest that PLD alpha is an important mediator in phytohormone-promoted senescence in detached leaves but is not a direct promoter of natural senescence. The physiological relevance of these findings is discussed.     

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D8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D8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。     

Regulation of plant water loss by manipulating the expression of phospholipase Dα

Phospholipase D (PLD) has been implicated in various processes, including signal transduction, membrane trafficking, and membrane degradation. Multiple forms of PLD with distinct biochemical properties have been described in the cell. In Arabidopsis, PLDalpha and PLDgamma, but not PLDbeta, were detected in guard cells, and antisense suppression resulted in a specific loss of PLDalpha. The abrogation of PLDalpha rendered plants less sensitive to abscisic acid and impaired stomatal closure induced by water deficits. PLDalpha-depleted plants exhibited accelerated transpirational water loss and a decreased ability to tolerate drought stress. Overexpression of PLDalpha enhanced the leaf's sensitivity to abscisic acid. These findings provide molecular and physiological evidence that PLDalpha plays a crucial role in regulating stomatal movement and plant-water status.     

Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis

A sensitive approach based on electrospray ionization tandem mass spectrometry has been employed to profile membrane lipid molecular species in Arabidopsis undergoing cold and freezing stresses. Freezing at a sublethal temperature induced a decline in many molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but induced an increase in phosphatidic acid (PA) and lysophospholipids. To probe the metabolic steps generating these changes, lipids of Arabidopsis deficient in the most abundant phospholipase D, PLD alpha, were analyzed. The PC content dropped only half as much, and PA levels rose only half as high in the PLD alpha-deficient plants as in wild-type plants. In contrast, neither PE nor PG levels decreased significantly more in wild-type plants than in PLD alpha-deficient plants. These data suggest that PC, rather than PE and PG, is the major in vivo substrate of PLD alpha. The action of PLD alpha during freezing is of special interest because Arabidopsis plants that are deficient in PLD alpha have improved tolerance to freezing. The greater loss of PC and increase in PA in wild-type plants as compared with PLD alpha-deficient plants may be responsible for destabilizing membrane bilayer structure, resulting in a greater propensity toward membrane fusion and cell death in wild-type plants.     

儿茶素诱导的拟南芥根细胞膜脂变化

儿茶素是一种可以短时间内杀死植物细胞的植物毒素,由于具有强的植物毒性,儿茶素是开发除草剂的理想化合物,它可以诱导植物根系统的死亡。为了研究植物根细胞膜脂对化学胁迫的响应规律,我们运用高通量的脂类组学方法检测了拟南芥根中膜脂分子的组成,比较了儿茶素处理下拟南芥野生型（WS）及磷脂酶Dδ缺失突变体（PLDδ KO）根中膜脂分子的组成情况、膜脂含量、双键指数及碳链长度值。结果发现,儿茶素处理拟南芥根90min后,二半乳糖基二酰甘油（DGDG）、单半乳糖基二酰甘油（MGDG）、磷脂酰甘油（PG）、磷脂酰胆碱（PC）及磷脂酰肌醇（PI）的总含量在WS与PLDδ KO植株根中都显著下降,磷脂酰乙醇胺（PE）和磷脂酰丝氨酸（PS）在WS中下降,在PLDδ KO中上升。儿茶素处理导致PLDδ KO植株的PC/PE比值显著下降,WS植株PS碳链长度显著增加。上述结果说明儿茶素处理后,磷脂酶Dδ缺失突变体膜不稳定性增加,PLDδ KO植株对儿茶素胁迫更加敏感。     

Overexpression of phospholipase D suppresses taxotere-induced cell death in stomach cancer cells

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid (PA) and choline. There are at least two PLD isozymes, PLD1 and PLD2. Genetic and pharmacological approaches implicate both PLD isozymes in a diverse range of cellular processes, including receptor signaling, membrane transport control, and actin cytoskeleton reorganization. Several recent studies reported that PLD has a role in signaling pathways that oppose apoptosis and promote cell survival in cancer. In this study, we examined the role of PLD in taxotere-induced apoptosis in stomach cell lines; normal stomach (NSC) and stomach cancer cells (SNU 484). Taxotere treatment resulted in increase of PLD activity. To confirm the role of PLD in taxotere-induced apoptosis, PLDs were transfected into SNU 484 cells. Overexpression of PLD isozymes resulted in inhibition of taxotere-induced apoptotic cell death, evidenced by decreased degradation of chromosomal DNA, and increased cell viability. Concurrently, Bcl-2 expression was upregulated, and taxotere-induced activation of procaspase 3 was inhibited after PLD's transfection. However, when PLD was selectively inhibited by specific siRNA-PLD1 or -PLD2, taxotere-induced apoptosis was exacerbated in SNU 484 cells. On top of this, PA -- the product of PLDs, also resulted in upregulation of Bcl-2 in SNU 484. Although PA-induced Bcl-2 expression was blocked by mepacrine, an inhibitor of phospholipase A(2) (PLA(2)), increased Bcl-2 expression by PA was not abrogated by propranolol, an inhibitor of PA phospholyhydrolase (PAP). Taken together, PLD1 and PLD2 are closely related with Bcl-2 expression together with PLA(2), but not with PAP, during taxotere-induced apoptosis in SNU 484 cells.     

The C-terminal domain of PLD2 participates in degradation of protein kinase CKII β subunit in human colorectal carcinoma cells

Elevated phospholipase D (PLD) expression prevents cell cycle arrest and apoptosis. However, the roles of PLD isoforms in cell proliferation and apoptosis are incompletely understood. Here, we investigated the physiological significance of the interaction between PLD2 and protein kinase CKII (CKII) in HCT116 human colorectal carcinoma cells. PLD2 interacted with the CKIIβ subunit in HCT116 cells. The C-terminal domain (residues 578-933) of PLD2 and the N-terminal domain of CKIIβ were necessary for interaction between the two proteins. PLD2 relocalized CKIIβ to the plasma membrane area. Overexpression of PLD2 reduced CKIIβ protein level, whereas knockdown of PLD2 led to an increase in CKIIβ expression. PLD2-induced CKIIβ reduction was mediated by ubiquitin-dependent degradation. The C-terminal domain of PLD2 was sufficient for CKIIβ degradation as the catalytic activity of PLD2 was not required. Taken together, the results indicate that the C-terminal domain of PLD2 can regulate CKII by accelerating CKIIβ degradation in HCT116 cells.     

Suppression of phospholipase Dγs confers increased aluminum resistance in Arabidopsis thaliana

Aluminum (Al) toxicity is the major stress in acidic soil that comprises about 50% of the world's arable land. The complex molecular mechanisms of Al toxicity have yet to be fully determined. As a barrier to Al entrance, plant cell membranes play essential roles in plant interaction with Al, and lipid composition and membrane integrity change significantly under Al stress. Here, we show that phospholipase Dγs (PLDγs) are induced by Al stress and contribute to Al-induced membrane lipid alterations. RNAi suppression of PLDγ resulted in a decrease in both PLDγ1 and PLDγ2 expression and an increase in Al resistance. Genetic disruption of PLDγ1 also led to an increased tolerance to Al while knockout of PLDγ2 did not. Both RNAi-suppressed and pldγ1-1 mutants displayed better root growth than wild-type under Al stress conditions, and PLDγ1-deficient plants had less accumulation of callose, less oxidative damage, and less lipid peroxidation compared to wild-type plants. Most phospholipids and glycolipids were altered in response to Al treatment of wild-type plants, whereas fewer changes in lipids occurred in response to Al stress in PLDγ mutant lines. Our results suggest that PLDγs play a role in membrane lipid modulation under Al stress and that high activities of PLDγs negatively modulate plant tolerance to Al.     

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.