麻疯树水通道蛋白新基因JcPIP干旱胁迫下的功能分析

采用RT-PCR和RACE技术,从大戟科(Euphorbiaceae)耐旱植物麻疯树(Jarropha curcas)cDNA中克隆得到了一个麻疯树质膜内膜蛋白(PIP)新基因,命名为JcPIP.聚类分析表明,麻疯树PIP蛋白与蓖麻、葡萄和菠菜的PIP蛋白在进化上有最近的亲缘关系.将JcPIP基因在非洲爪蟾卵母细胞(xenopus oocytes)中异源表达发现细胞膨胀率扩大了10倍,表明JePIP基因编码的是一个水通道蛋白.合成亲水性、抗原性好的JcPIP保守序列多肽,制备并纯化JcPIP多克隆抗体.Western-bbt检测显示JcPIP蛋白富集在29 kDa区段,且在麻疯树各组织中均有大量表达.PEG-6000干旱胁迫下麻疯树叶片JcPIP蛋白丰度增加,复水后丰度开始下降,表明JcPlP与麻疯树的耐旱性相关.     

Drought, abscisic acid and transpiration rate effects on the regulation of PIP aquaporin gene expression and abundance in Phaseolus vulgaris plants

BACKGROUND AND AIMS: Drought causes a decline of root hydraulic conductance, which aside from embolisms, is governed ultimately by aquaporins. Multiple factors probably regulate aquaporin expression, abundance and activity in leaf and root tissues during drought; among these are the leaf transpiration rate, leaf water status, abscisic acid (ABA) and soil water content. Here a study is made of how these factors could influence the response of aquaporin to drought. METHODS: Three plasma membrane intrinsic proteins (PIPs) or aquaporins were cloned from Phaseolus vulgaris plants and their expression was analysed after 4 d of water deprivation and also 1 d after re-watering. The effects of ABA and of methotrexate (MTX), an inhibitor of stomatal opening, on gene expression and protein abundance were also analysed. Protein abundance was examined using antibodies against PIP1 and PIP2 aquaporins. At the same time, root hydraulic conductance (L), transpiration rate, leaf water status and ABA tissue concentration were measured. KEY RESULTS: None of the treatments (drought, ABA or MTX) changed the leaf water status or tissue ABA concentration. The three treatments caused a decline in the transpiration rate and raised PVPIP2;1 gene expression and PIP1 protein abundance in the leaves. In the roots, only the drought treatment raised the expression of the three PIP genes examined, while at the same time diminishing PIP2 protein abundance and L. On the other hand, ABA raised both root PIP1 protein abundance and L. CONCLUSIONS: The rise of PvPIP2;1 gene expression and PIP1 protein abundance in the leaves of P. vulgaris plants subjected to drought was correlated with a decline in the transpiration rate. At the same time, the increase in the expression of the three PIP genes examined caused by drought and the decline of PIP2 protein abundance in the root tissues were not correlated with any of the parameters measured.     

Plasma membrane intrinsic proteins from maize cluster in two sequence subgroups with differential aquaporin activity

The transport of water through membranes is regulated in part by aquaporins or water channel proteins. These proteins are members of the larger family of major intrinsic proteins (MIPs). Plant aquaporins are categorized as either tonoplast intrinsic proteins (TIPs) or plasma membrane intrinsic proteins (PIPs). Sequence analysis shows that PIPs form several subclasses. We report on the characterization of three maize (Zea mays) PIPs belonging to the PIP1 and PIP2 subfamilies (ZmPIP1a, ZmPIP1b, and ZmPIP2a). The ZmPIP2a clone has normal aquaporin activity in Xenopus laevis oocytes. ZmPIP1a and ZmPIP1b have no activity, and a review of the literature shows that most PIP1 proteins identified in other plants have no or very low activity in oocytes. Arabidopsis PIP1 proteins are the only exception. Control experiments show that this lack of activity of maize PIP1 proteins is not caused by their failure to arrive at the plasma membrane of the oocytes. ZmPIP1b also does not appear to facilitate the transport of any of the small solutes tried (glycerol, choline, ethanol, urea, and amino acids). These results are discussed in relationship to the function and regulation of the PIP family of aquaporins.     

水分亏缺下玉米根系ZmPIP1亚族基因的表达

在PEG-6000胁迫条件下,以微管蛋白基因为内参基因、水通道蛋白基因ZmPIP1-1和ZmPIP1-2为检测基因,采用半定量逆转录聚合酶链式反应（RT-PCR）体系检测它们在玉米根系中的表达情况。实验结果是：胁迫条件下,ZmPIP1-1的表达量在杂交F,代‘户单4号’（抗旱）和母本‘天四’（抗旱）根系中增多,它的表达量与品种的抗旱性呈正相关,并且胁迫不同时间段它的表达量有差异;而ZmPIP1-2在3个玉米品种的不同水分处理条件下,表达量均没有明显变化。这提示,水分胁迫条件下根系中某些种类的水通道蛋白基因的表达量增多,并且与品种的抗旱性有关;而另一些水通道蛋白基因的表达不受水分亏缺的影响。     

干旱胁迫对不同施氮水平麻疯树幼苗光合特性及生长的影响

采用盆栽控水的方法,研究了干旱胁迫（连续干旱0 d,5 d,10 d,…,45 d）对不同施氮水平(对照 0 kg N·hm^-2、低氮 96 kg N·hm^-2、中氮 288 kg N·hm^-2、高氮 480 kg N·hm^-2)麻疯树幼苗光合特性及其生长的影响.结果表明: 随干旱胁迫强度的增加,各施氮水平麻疯树幼苗叶片相对含水量、苗高生长量、地径生长量、叶面积、净光合速率、蒸腾速率和气孔导度均降低,且各水分处理间差异极显著(P<0.01);随干旱时间的延长,叶绿素含量和水分利用效率表现出先升高后降低的趋势,而胞间CO₂浓度呈先降低后升高的趋势.正常供水时,施氮处理均不同程度提高了麻疯树幼苗的光合能力,促进了麻疯树幼苗的生长,且施氮量越高效果越好;干旱条件下,氮素营养对植株光合能力和生长的影响与干旱程度和施氮水平有关.轻度干旱时,提高施氮水平对植株光合能力和生长具有明显的促进作用;中度干旱时,中氮的促进作用明显高于其他施氮水平;严重干旱时,低氮的促进效果最好,高氮的促进作用减弱并逐渐转向抑制.     

Water deficit during root development: effects on the growth of roots and osmotic water permeability of isolated root protoplasts

The effect of low water potentials on root growth of flax (Linum usitatissimum L. cv. Ariane), rape (Brassica napus L. de Candolle, cv. Bristol), hard wheat (Triticum turgidum L. cv. Cham1) and soft wheat (Triticum aestivum L. cv. Ritmo) was studied by measuring the osmotic water permeability (Pos) of root protoplasts and the protein abundance of PIP1 and PIP2 aquaporins. These different species require more or less water, the most sensitive to water deficit being flax and rape. Ritmo, is a cultivar of wheat adapted to temperate zones, while the other cultivar Cham1 is adapted to low-rainfall areas. The seedlings were germinated and grown in water, salt or sugar solutions at different water potentials. The values of Pos for flax, rape and Chaml wheat were normally distributed and could be characterized by mean +/- SD. Root protoplasts from water-grown seedlings had Pos values of 485+/-159 microm s(-1) (flax), 582+/-100 microm s(-1) (rape), and 6.3+/-3.5 microm s(-1) (Cham1). At the same age, the protoplasts from Ritmo exhibited a much wider range of values than the protoplasts of Cham1. When seedlings were grown under conditions of osmotic or salt stress, the mass of the roots was reduced for all species. With 0.25 mol kg(-1) sorbitol or 0.125 M NaCl, the Pos for flax, rape and Cham1 remained constant or slightly increased, while for Ritmo the reduction in the mass of the roots was paralleled by a reduction in Pos. Only Cham1 and Ritmo were able to germinate at a lower potential (0.5 mol kg(-1) sorbitol). For Ritmo the reduction in the mass of the roots was paralleled by a reduction in Pos when grown in this stress condition and both wheats exhibited low Pos values. The expression of the PIP1 and PIP2 aquaporins families was also studied by immunoblotting. We did not observe any difference in protein expression for any of the species, whatever the growing conditions. We suggest that the high Pos values for flax and rape could play a role in the sensitivity of these plants at low water potential. The low native Pos for Cham1 in spite of the expression of both families of aquaporins may reflect its adaptation to low-rainfall conditions by a functional regulation of the water channels. For a similar reason, the low-water-potential-induced Pos of Ritmo may also correspond to a down-regulation of the aquaporins, reflecting adaptation of this wheat to water-deficit conditions.     

赤霉素、钙和甜菜碱对小桐子种子萌发及幼苗抗低温和干旱的影响

小桐子(Jatropha curcas L.)属大戟科( Euphorbiaceae)麻疯树属(Jatropha L.)的能源植物。分别以小桐子种子和幼苗为实验材料,研究了不同浓度的赤霉素和CaCl₂单独处理,以及不同浓度的赤霉素、CaCl₂和甜菜碱组合处理对小桐子种子萌发及幼苗抗低温和干旱的影响。结果表明:分别用10 mg/L赤霉素和10 mmol/L CaCl₂处理小桐子种子,不仅可以提高其在正常萌发条件(26℃)、低温(18℃)和干旱胁迫(5% PEG6000)下的发芽率,还可缓解小桐子幼苗在低温(2℃)或干旱胁迫(25% PEG6000)下电解质渗漏率的增加和丙二醛(MDA)的积累。用10 mg/L赤霉素、5 mmol/L CaCl₂和15 mmol/L甜菜碱组合处理小桐子种子,也可进一步增强其在正常萌发(26℃)、低温(18℃)和干旱胁迫(5% PEG6000)条件下种子的发芽率,以及缓解小桐子幼苗在低温(2℃)或干旱胁迫(25% PEG6000)下电解质渗漏率的增加和MDA的积累,表明10 mg/L赤霉素和10 mmol/L CaCl₂分别处理或10 mg/L赤霉素、5 mmol/L CaCl₂和10 mmol/L甜菜碱组合处理可提高小桐子种子在低温和干旱胁迫下的发芽率,以及提高小桐子幼苗对低温和干旱胁迫的抵抗能力。     

Plasma membrane aquaporins are involved in winter embolism recovery in walnut tree

In perennial plants, freeze-thaw cycles during the winter months can induce the formation of air bubbles in xylem vessels, leading to changes in their hydraulic conductivity. Refilling of embolized xylem vessels requires an osmotic force that is created by the accumulation of soluble sugars in the vessels. Low water potential leads to water movement from the parenchyma cells into the xylem vessels. The water flux gives rise to a positive pressure essential for the recovery of xylem hydraulic conductivity. We investigated the possible role of plasma membrane aquaporins in winter embolism recovery in walnut (Juglans regia). First, we established that xylem parenchyma starch is converted to sucrose in the winter months. Then, from a xylem-derived cDNA library, we isolated two PIP2 aquaporin genes (JrPIP2,1 and JrPIP2,2) that encode nearly identical proteins. The water channel activity of the JrPIP2,1 protein was demonstrated by its expression in Xenopus laevis oocytes. The expression of the two PIP2 isoforms was investigated throughout the autumn-winter period. In the winter period, high levels of PIP2 mRNA and corresponding protein occurred simultaneously with the rise in sucrose. Furthermore, immunolocalization studies in the winter period show that PIP2 aquaporins were mainly localized in vessel-associated cells, which play a major role in controlling solute flux between parenchyma cells and xylem vessels. Taken together, our data suggest that PIP2 aquaporins could play a role in water transport between xylem parenchyma cells and embolized vessels.     

Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress

Most of the symplastic water transport in plants occurs via aquaporins, but the extent to which aquaporins contribute to plant water status under favorable growth conditions and abiotic stress is not clear. To address this issue, we constitutively overexpressed the Arabidopsis plasma membrane aquaporin, PIP1b, in transgenic tobacco plants. Under favorable growth conditions, PIP1b overexpression significantly increased plant growth rate, transpiration rate, stomatal density, and photosynthetic efficiency. By contrast, PIP1b overexpression had no beneficial effect under salt stress, whereas during drought stress it had a negative effect, causing faster wilting. Our results suggest that symplastic water transport via plasma membrane aquaporins represents a limiting factor for plant growth and vigor under favorable conditions and that even fully irrigated plants face limited water transportation. By contrast, enhanced symplastic water transport via plasma membrane aquaporins may not have any beneficial effect under salt stress, and it has a deleterious effect during drought stress.     

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins （PIPs）, was studied at both the protein and mRNA level in upland rice （Oryza sativa L. cv. Zhonghan 3） and lowland rice （Oryza sativa L. cv. Xiushui 63） when they were water stressed by treatment with 20% polyethylene glycol （PEG）. Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1,2, OsPIP1,3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1,2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid （ABA） level in upland rice but not in lowland rice under water deficit. Treatment with 60 μM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling oathways during water deficit.     

Methylation of aquaporins in plant plasma membrane

A thorough analysis, using MS, of aquaporins expressed in plant root PM (plasma membrane) was performed, with the objective of revealing novel post-translational regulations. Here we show that the N-terminal tail of PIP (PM intrinsic protein) aquaporins can exhibit multiple modifications and is differentially processed between members of the PIP1 and PIP2 subclasses. Thus the initiating methionine was acetylated or cleaved in native PIP1 and PIP2 isoforms respectively. In addition, several residues were detected to be methylated in PIP2 aquaporins. Lys3 and Glu6 of PIP2;1, one of the most abundant aquaporins in the PM, occurred as di- and mono-methylated residues respectively. Ectopic expression in Arabidopsis suspension cells of PIP2;1, either wild-type or with altered methylation sites, revealed an interplay between methylation at the two sites. Measurements of water transport in PM vesicles purified from these cells suggested that PIP2;1 methylation does not interfere with the aquaporin intrinsic water permeability. In conclusion, the present study identifies methylation as a novel post-translational modification of aquaporins, and even plant membrane proteins, and may represent a critical advance towards the identification of new regulatory mechanisms of membrane transport.     

Significance of plasmalemma aquaporins for water‐transport in Arabidopsis thaliana

The plant plasma membrane intrinsic protein, PIP1b, facilitates water transport. These features were characterized in Xenopus oocytes and it has asked whether aquaporins are relevant for water transport in plants. In order to elucidate this uncertainty Arabidopsis thaliana was transformed with an anti-sense construct targeted to the PIP1b gene. Molecular analysis revealed that the anti-sense lines have reduced steady-state levels of PIP1b and the highly homologous PIP1a mRNA. The cell membrane water permeability was analyzed by swelling of protoplasts, which had been transferred into hypotonic conditions. The results indicate that the reduced expression of the specific aquaporins decreases the cellular osmotic water permeability coefficient approximately three times. The morphology and development of the anti-sense lines resembles that of control plants, with the exception of the root system, which is five times as abundant as that of control plants. Xylem pressure measurement suggests that the increase of root mass compensates the reduced cellular water permeability in order to ensure a sufficient water supply to the plant. The results obtained by this study, therefore, clearly demonstrate that aquaporins are important for plant water transport.     

PIP1 and PIP2 aquaporins are differentially expressed during tobacco anther and stigma development

Several processes during sexual reproduction in higher plants involve the movement of water between cells or tissues, such as occurs during dehiscence of the anther and hydration of the pollen grain after it is deposited on a stigma. To get more insight in these processes, a set of putative aquaporins was cloned and it was found that at least 15 are expressed in reproductive organs, which indicates that the control of water flow is important for reproduction. Functional studies in Xenopus laevis oocytes using two of the cDNAs showed that NtPIP2;1 is an efficient aquaporin, whereas NtPIP1;1 is not. Expression studies on RNA and protein levels showed that PIP1 and PIP2 genes are differently expressed in reproductive organs: PIP1 RNA accumulates in the stigma, and PIP1 and PIP2 RNA can be detected in most tissues of the anther.     

MzPIP2;1: An Aquaporin Involved in Radial Water Movement in Both Water Uptake and Transportation,Altered the Drought and Salt Tolerance of Transgenic Arabidopsis

Background

Plants are unavoidably subjected to various abiotic stressors, including high salinity, drought and low temperature, which results in water deficit and even death. Water uptake and transportation play a critical role in response to these stresses. Many aquaporin proteins, localized at different tissues, function in various transmembrane water movements. We targeted at the key aquaporin in charge of both water uptake in roots and radial water transportation from vascular tissues through the whole plant.

Results

The MzPIP2;1 gene encoding a plasma membrane intrinsic protein was cloned from salt-tolerant apple rootstock Malus zumi Mats. The GUS gene was driven by MzPIP2;1 promoter in transgenic Arabidopsis. It indicated that MzPIP2;1 might function in the epidermal and vascular cells of roots, parenchyma cells around vessels through the stems and vascular tissues of leaves. The ectopically expressed MzPIP2;1 conferred the transgenic Arabidopsis plants enhanced tolerance to slight salt and drought stresses, but sensitive to moderate salt stress, which was indicated by root length, lateral root number, fresh weight and K⁺/Na⁺ ratio. In addition, the possible key cis-elements in response to salt, drought and cold stresses were isolated by the promoter deletion experiment.

Conclusion

The MzPIP2;1 protein, as a PIP2 aquaporins subgroup member, involved in radial water movement, controls water absorption and usage efficiency and alters transgenic plants drought and salt tolerance.