植物抗逆反应中水孔蛋白的表达调控研究

水孔蛋白(aquaporin,AQP)作为一种功能性跨膜输水蛋白,对植物体形成水选择性运输通道并实现水分的跨膜运输起到重要的作用.当植物处于盐、干旱、低温等逆境胁迫状态时,体内的水分平衡被打破,水孔蛋白在水分运输和胞内渗透压的调控等方面表现出重要作用.综述了植物抗逆反应中水孔蛋白表达调控的研究进展.     

荒漠绿洲过渡带一年生草本植物对干旱胁迫的响应

选取河西走廊荒漠绿洲过渡带典型一年生草本植物雾冰藜（Bassia dasyphylla）、虎尾草（Chloris virgata）和狗尾草（Setaria viridis）为研究对象,设置5个水分梯度（正常水分（CK）,轻度干旱（5d）、中度干旱（10d）、重度干旱（15d）,重度干旱（15d）复水）,分析了3种一年生草本植物生理和形态等性状对干旱胁迫的响应。结果表明：一年生草本植物可以通过生理反应（渗透调节）适应轻度和中度干旱胁迫,而通过个体形态来适应重度干旱胁迫。在轻度和中度干旱处理下,一年生草本植物通过调控叶片渗透调节物质脯氨酸、可溶性蛋白和可溶性糖维持叶片渗透压,提高保水能力,叶绿素含量增加,使丙二醛含量维持在较低的水平,同时,根系活力增强,有效促进了根系水分吸收;而在重度胁迫下,渗透调节物质作用降低,丙二醛含量迅速增加,导致可溶性蛋白含量下降,叶绿素分解加速,植物生长受到抑制,在有限的生物量下,一年生草本植物主要通过根系伸长、根长与茎长的比增加和减小茎长来适应重度干旱胁迫,最终导致了种子百粒重和结种数量下降。     

植物水通道蛋白的干旱应答机制研究进展

干旱胁迫是严重影响全球作物生产的非生物胁迫之一,研究植物耐旱机制已成为一个重要领域。水通道蛋白是一类特异、高效转运水及其它小分子底物的膜通道蛋白,在植物中具有丰富的亚型,参与调节植物的水分吸收和运输。近10年来,水通道蛋白在植物不同生理过程中的作用,一直受到研究人员的关注,特别是在非生物胁迫方面,而研究表明水通道蛋白在干旱胁迫下对植物的耐旱性起着至关重要的作用,能维持细胞水分稳态和调控环境胁迫快速响应。水通道蛋白在植物耐旱过程中的调控机制及功能较复杂,而关于其应答机制和不同亚型功能性研究的报道甚少。该文综述了植物水通道蛋白的分类、结构、表达调控和活性调节,分别从植物水通道蛋白响应干旱表达调控机制、水通道蛋白基因表达的时空特异性、水通道蛋白基因的表达与蛋白丰度,水通道蛋白基因的耐旱转化四个方面阐明干旱胁迫下植物水通道蛋白的表达,重点阐述其参与植物干旱胁迫应答的作用机制,并提出水通道蛋白研究的主要方向。     

干旱胁迫诱导下植物基因的表达与调控

干旱胁迫能够诱导植物表达大量的基因 ,研究这些基因的表达与调控 ,为植物抗旱的定向育种创造条件。本文系统介绍了在干旱胁迫条件下 ,植物体内渗透调节物质和可溶性糖合成有关的基因、离子和水分通道及Lea蛋白基因的表达 ,以及与这些基因表达相关的调控元件和因子 ,干旱胁迫信号转导等方面的最新研究进展。     

利用烟草瞬时表达系统检测高等植物基因的剪接加工

解析基因的剪接加工机制是了解植物形态建成、生长发育和逆境胁迫应答的重要环节．与动物相比,植物中相应的研究进展较为缓慢．利用农杆菌介导的烟草瞬时表达系统,分别对单子叶植物水稻BADH2和双子叶植物拟南芥GR7基因片段在烟草叶片中的转录后剪接加工进行分析．结果表明,一些重要剪接调控元件在植物中保守存在,而烟草瞬时表达系统可以作为研究高等植物剪接调控的重要工具,快捷灵敏地检测基因的剪接加工方式．     

22种植物水孔蛋白理化性质及其结构特征的生物信息学分析

水孔蛋白(aquaporins, AQPs)是高效转运水分子的膜内在蛋白,具有丰富的多样性,在调控植物的水分关系中有重要作用。本研究采用Blast、ProtParam、SignalP和Swiss-Model等生物信息学软件,对梨、苹果、黄瓜和番茄等22种植物AQP的理化性质、蛋白结构、系统发生树和功能域等进行了分析。结果表明, AQP蛋白氨基酸长度在284~295 aa之间,理论等电点在7.67~9.30之间,主要定位于质膜上,除红叶藜外均为稳定性蛋白。二级结构由α螺旋、β折叠、无规则卷曲和延伸链等结构元件组成,空间结构高度相似,属于主要内在蛋白(MIP)家族。     

植物体内钙信号及其在调节干旱胁迫中的作用

钙作为植物体内第二信使广泛参与了植物响应的各种非生物和生物胁迫的信号传导。胁迫信号通过激活位于细胞质膜上的钙离子通道,产生胞质内特异性的钙信号,传递至钙信号感受蛋白,如钙调素(calmodulin,CaM)、钙依赖蛋白激酶(Ca2+-dependent protein kinases,CDPK)和类钙调磷酸酶B蛋白(calcineurin B-like protein,CBL)等,进而引起胞内一系列生理生化变化,最终对胁迫做出响应。钙信号在植物响应干旱胁迫信号系统中起枢纽作用,主要通过调节气孔运动,水通道蛋白(aquaporin,AQP)和抗氧化酶活性来减少水分流失,提高水分利用率,最终降低干旱对植物细胞的伤害,并具有一定的生态学功能。该文对近年来国内外有关植物体内钙信号的研究进展以及在干旱逆境中的调节作用进行综述,并对今后的研究做了展望。     

垫状卷柏SpLEA1基因克隆及干旱胁迫下的表达分析

晚期胚胎发育丰富蛋白(late embryogenesis abundant,LEA),广泛存在于生物体内,与植物抗逆性密切相关,可在干旱胁迫下保护植物细胞,减少植物损伤。垫状卷柏(Selaginella pulvinata)是一种在干旱胁迫下生存能力极强的蕨类植物,具有很强的恢复能力。为探究垫状卷柏SpLEA1基因在耐旱植物中的分子机制与表达特征,该研究以高耐旱性植物垫状卷柏为实验材料,基于转录组测序结果,采用RT-PCR技术获得SpLEA1基因cDNA序列,采用HiTail-PCR技术获得启动子序列,利用生物信息学对序列进行了分析,并采用qRT-PCR技术分析了SpLEA1基因在干旱胁迫下的表达模式。结果表明:(1)垫状卷柏SpLEA1全长为476 bp,开放阅读框(ORF)为279 bp,共编码92个氨基酸,通过在线工具预测到蛋白分子量为9 491.46 Da, 等电点为5.45,蛋白结构预测分析表明该蛋白为亲水性蛋白,含有10个磷酸化位点,二级结构以α-螺旋和无规则卷曲为主。(2)预测到SpLEA1蛋白的保守结构域为Lea-5,来源于LEA1家族。基于系统发生树和遗传距离矩阵,发现垫状卷柏SpLEA1与来自鹰嘴豆(Cicer arietinum )和红车轴草(Trifolium pratense)的Lea-5蛋白同源性较高。(3)对启动子序列进行顺式作用元件的预测分析发现SpLEA1基因启动子含有5类激素响应元件和与干旱胁迫响应有关的功能元件。(4)在自然干旱处理下SpLEA1基因表达上调,并在12 h时达到峰值,在24 h干旱后进行复水处理,表达量显著下调。综上所述,SpLEA1基因在垫状卷柏中很可能参与了干旱胁迫响应机制的相关调控。该结果为进一步研究垫状卷柏SpLEA1基因在干旱胁迫下的功能及其表达调控机制提供了参考。     

蛋白质组学在植物水分胁迫应答中的应用研究进展

水分胁迫是影响植物生长发育的主要生长因子。通过蛋白质组学技术可对水分胁迫下植物差异变化的蛋白和基因进行挖掘,在研究植物抗旱生理机制方面意义重大。总结了植物蛋白质组学的基本方法与关键技术,同时从光合与碳代谢相关蛋白、抗氧化系统、渗透调节蛋白、热激蛋白、胚胎发育晚期丰富蛋白、转录因子等方面综述了近几年国际上在植物水分胁迫蛋白质组研究方面的进展,并展望了今后蛋白质组学技术发展的方向。     

沙蒿AQP基因结构预测及干旱胁迫下的时空表达模式研究

为探究水通道蛋白(AQP)在沙蒿响应干旱胁迫中的作用机制,该研究以青海省柴达木盆地沙蒿为试验材料,采用RACE技术对其AQP基因进行扩增,获得沙蒿AQP全长克隆并对AQP蛋白进行结构预测和分析;采用qRT-PCR对沙蒿AQP基因在不同程度干旱胁迫以及不同组织部位的表达模式进行分析。结果表明:(1)成功克隆获得沙蒿AQP基因长746 bp的片段1和长534 bp的片段2,经拼接后得到全长cDNA序列,沙蒿AQP基因总长为864 bp。(2)亚细胞定位表明沙蒿AQP基因定位于细胞膜上;同源比对显示沙蒿与向日葵、莴苣、橡胶树等植物的AQP基因具有较高的相似性;结构预测表明AQP蛋白含6个跨膜螺旋结构且亲水性较弱,α螺旋和无规则卷曲为AQP蛋白二级结构的主要构成元件。(3)qRT-PCR分析表明,沙蒿AQP基因随着干旱胁迫的加重呈现有规律的变化,根、茎、叶中表达均上调,且叶中AQP基因表达量上调幅度最大。研究表明沙蒿AQP基因结构特征及其表达模式都是沙蒿对干旱胁迫的一种适应。     

Low-level laser therapy with 850 nm recovers salivary function via membrane redistribution of aquaporin 5 by reducing intracellular Ca2+ overload and ER stress during hyperglycemia

The overall goal is to study the effect of low-level laser therapy (LLLT) on membrane distribution of major water channel protein aquaporin 5 (AQP5) in salivary gland during hyperglycemia. Par C10 cells treated with high glucose (50?mM) showed a reduced membrane distribution of AQP5. The functional expression of AQP5 was downregulated due to intracellular Ca²⁺ overload and ER stress. This reduction in AQP5 expression impairs water permeability and therefore results in hypo-salivation. A reduced salivary flow was also observed in streptozotocin (STZ)-induced diabetic mice model and the expression of AQP5 and phospho-AQP5 was downregulated. Low-level laser treatment with 850?nm (30?mW, 10?min?=?18?J/cm²) reduced ER stress and recovered AQP5 membrane distribution via serine phosphorylation in the cells. In the STZ-induced diabetic mouse, LLLT with 850?nm (60?J/cm²) increased salivary flow and upregulated of AQP5 and p-AQP5. ER stress was also reduced via downregulation of caspase 12 and CHOP. In silico analysis confirmed that the serine 156 is one of the most favorable phosphorylation sites of AQP5 and may contribute to the stability of the protein. Therefore, this study suggests high glucose inhibits phosphorylation-dependent AQP5 membrane distribution. High glucose induces intracellular Ca²⁺ overload and ER stress that disrupt AQP5 functional expression. Low-level laser therapy with 850?nm improves salivary function by increasing AQP5 membrane distribution in hyperglycemia-induced hyposalivation.     

Hypertonic induction of aquaporin-5 expression through an ERK-dependent pathway

Aquaporin-5 (AQP5) is a water channel protein expressed in lung, salivary gland, and lacrimal gland epithelia. Each of these sites may experience fluctuations in surface liquid osmolarity; however, osmotic regulation of AQP5 expression has not been reported. This study demonstrates that AQP5 is induced by hypertonic stress and that induction requires activation of extracellular signal-regulated kinase (ERK). Incubation of mouse lung epithelial cells (MLE-15) in hypertonic medium produced a dose-dependent increase in AQP5 expression; AQP5 protein peaked by 24 h and returned to baseline levels within hours of returning cells to isotonic medium. AQP5 induction was observed only with relatively impermeable solutes, suggesting an osmotic pressure gradient is required for induction. ERK was selectively activated in MLE-15 cells by hypertonic stress, and inhibition of ERK activation with two distinct mitogen-activated extracellular regulated kinase kinase (MEK) inhibitors, U0126 and PD98059, blocked AQP5 induction. AQP5 induction was also observed in the lung, salivary, and lacrimal glands of hyperosmolar rats, suggesting potential physiologic relevance for osmotic regulation of AQP5 expression. This report provides the first example of hypertonic induction of an extrarenal aquaporin, as well as the first association between mitogen-activated protein kinase signaling and aquaporin expression.     

Glutathionylation of the Aquaporin-2 Water Channel: A NOVEL POST-TRANSLATIONAL MODIFICATION MODULATED BY THE OXIDATIVE STRESS*

Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and urine concentration. AQP2 undergoes different regulated post-translational modifications, including phosphorylation and ubiquitylation, which are fundamental for controlling AQP2 cellular localization, stability, and function. The relationship between AQP2 and S-glutathionylation is of potential interest because reactive oxygen species (ROS), produced under renal failure or nephrotoxic drugs, may influence renal function as well as the expression and the activity of different transporters and channels, including aquaporins. Here, we show for the first time that AQP2 is subjected to S-glutathionylation in kidney and in HEK-293 cells stably expressing AQP2. S-Glutathionylation is a redox-dependent post-translational modification controlling several signal transduction pathways and displaying an acute effect on free cytosolic calcium concentration. Interestingly, we found that in fresh kidney slices, the increased AQP2 S-glutathionylation correlated with tert-butyl hydroperoxide-induced ROS generation. Moreover, we also found that cells expressing wild-type human calcium-sensing receptor (hCaSR-wt) and its gain of function (hCaSR-R990G; hCaSR-N124K) had a significant decrease in AQP2 S-glutathionylation secondary to reduced ROS levels and reduced basal intracellular calcium concentration compared with mock cells. Together, these new findings provide fundamental insight into cell biological aspects of AQP2 function and may be relevant to better understand and explain pathological states characterized by an oxidative stress and AQP2-dependent water reabsorption disturbs.     

Lung edema clearance: 20 years of progress: invited review: role of aquaporin water channels in fluid transport in lung and airways.

Water transport across epithelial and endothelial barriers in bronchopulmonary tissues occurs during airway hydration, alveolar fluid transport, and submucosal gland secretion. Many of the tissues involved in these processes are highly water permeable and express aquaporin (AQP) water channels. AQP1 is expressed in microvascular endothelia throughout the lung and airways, AQP3 in epithelia in large airways, AQP4 in epithelia throughout the airways, and AQP5 in type I alveolar epithelial cells and submucosal gland acinar cells. The expression of some of these AQPs increases near the time of birth and is regulated by growth factors, inflammation, and osmotic stress. Transgenic mouse models of AQP deletion have provided information about their physiological role. In lung, AQP1 and AQP5 provide the principal route for osmotically driven water transport; however, alveolar fluid clearance in the neonatal and adult lung is not affected by AQP deletion nor is lung CO(2) transport or fluid accumulation in experimental models of lung injury. In the airways, AQP3 and AQP4 facilitate water transport; however, airway hydration, regulation of the airway surface liquid layer, and isosmolar fluid absorption are not impaired by AQP deletion. In contrast to these negative findings, AQP5 deletion in submucosal glands in upper airways reduced fluid secretion and increased protein content by greater than twofold. Thus, although AQPs play a major physiological role outside of the airways and lung, AQPs appear to be important mainly in airway submucosal gland function. The substantially slower rates of fluid transport in airways, pleura, and lung compared with renal and some secretory epithelia may account for the apparent lack of functional significance of AQPs at these sites. However, the possibility remains that AQPs may play a role in lung physiology under conditions of stress and/or injury not yet tested or in functions unrelated to transepithelial fluid transport.     

An inhibitory role of Rho in the vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells

Vasopressin regulates water reabsorption in renal collecting duct principal cells by a cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the cell membrane. In the present work primary cultured inner medullary collecting duct cells were used to study the role of the proteins of the Rho family in the translocation of AQP2. Clostridium difficile toxin B, which inhibits all members of the Rho family, Clostridium limosum C3 toxin, which inactivates only Rho, and the Rho kinase inhibitor, Y-27632, induced both depolymerization of actin stress fibers and AQP2 translocation in the absence of vasopressin. The data suggest an inhibitory role of Rho in this process, whereby constitutive membrane localization is prevented in resting cells. Expression of constitutively active RhoA induced formation of actin stress fibers and abolished AQP2 translocation in response to elevation of intracellular cAMP, confirming the inhibitory role of Rho. Cytochalasin D induced both depolymerization of the F-actin cytoskeleton and AQP2 translocation, indicating that depolymerization of F-actin is sufficient to induce AQP2 translocation. Thus Rho is likely to control the intracellular localization of AQP2 via regulation of the F-actin cytoskeleton.     

Hyperosmolar mannitol simulates expression of aquaporins 4 and 9 through a p38 mitogen-activated protein kinase-dependent pathway in rat astrocytes

The membrane pore proteins, aquaporins (AQPs), facilitate the osmotically driven passage of water and, in some instances, small solutes. Under hyperosmotic conditions, the expression of some AQPs changes, and some studies have shown that the expression of AQP1 and AQP5 is regulated by MAPKs. However, the mechanisms regulating the expression of AQP4 and AQP9 induced by hyperosmotic stress are poorly understood. In this study, we observed that hyperosmotic stress induced by mannitol increased the expression of AQP4 and AQP9 in cultured rat astrocytes, and intraperitoneal infusion of mannitol increased AQP4 and AQP9 in the rat brain cortex. In addition, a p38 MAPK inhibitor, but not ERK and JNK inhibitors, suppressed their expression in cultured astrocytes. AQPs play important roles in maintaining brain homeostasis. The expression of AQP4 and AQP9 in astrocytes changes after brain ischemia or traumatic injury, and some studies have shown that p38 MAPK in astrocytes is activated under similar conditions. Since mannitol is commonly used to reduce brain edema, understanding the regulation of AQPs and p38 MAPK in astrocytes under hyperosmotic conditions induced with mannitol may lead to a control of water movements and a new treatment for brain edema.