刚毛柽柳NAC24基因的表达及抗逆功能分析

【目的】NAC类转录因子是植物特有的最大的转录因子家族之一,广泛参与植物的生长发育过程,并在植物响应盐、干旱等多种非生物胁迫的过程中发挥至关重要的调控作用。本研究拟从盐生木本植物刚毛柽柳中克隆获得一个NAC转录因子基因,研究该基因的耐盐、抗旱功能,以期为研究木本植物NAC转录因子的抗逆分子机制奠定理论基础。【方法】在刚毛柽柳NaHCO_3胁迫转录组数据库中筛选获得一个NAC转录因子基因,将其命名为ThNAC24(GenBank登陆号:KF031949)。利用生物信息学工具将其与其他9个物种的NAC蛋白进行多序列比对,与拟南芥105个NAC蛋白进行进化树分析。分别用300 mmol·L^-1 NaCl和400 mmol·L^-1甘露醇对刚毛柽柳进行胁迫,在胁迫6、12、24和48 h后分别取刚毛柽柳根及叶组织。通过实时荧光定量RT-PCR(qRT-PCR)技术分析盐、干旱胁迫下ThNAC24基因在不同胁迫时间点及不同组织的表达情况,初步鉴定其是否响应盐、干旱胁迫。为进一步研究ThNAC24基因的抗逆功能,分别构建植物过表达(pROKⅡ-ThNAC24)及抑制表达(pFGC5941-ThNAC24)载体。利用农杆菌介导的高效瞬时遗传转化体系获得ThNAC24基因瞬时过表达(OE)、抑制表达(IE)及对照(Control)刚毛柽柳植株。在盐、干旱胁迫下分析比较了ThNAC24基因瞬时过表达、抑制表达及对照刚毛柽柳植株的二氨基联苯胺(DAB)和氯化硝基四氮唑蓝(NBT)染色情况,过氧化物酶(POD)和超氧化物歧化酶(SOD)活性,及电解质渗透率、失水率及丙二醛(MDA)含量,鉴定ThNAC24基因的耐盐、抗旱功能。【结果】ThNAC24基因的开放阅读框为1 023 bp,编码340个氨基酸。多序列比对结果显示ThNAC24在N端的氨基酸序列相似度比较高,具有NAC家族的序列特征;系统进化树分析结果显示ThNAC24与ANAC103和ANAC082的亲缘关系较近。qRT-PCR结果显示:盐胁迫下,ThNAC24基因上调表达,在根组织中胁迫12 h表达量最高,而叶组织中胁迫24 h的表达量最高;干旱胁迫下,ThNAC24基因上调表达,在根组织中胁迫6 h表达量最高,在叶组织中胁迫12 h的表达量最高。ThNAC24基因在刚毛柽柳根和叶组织中均有表达且响应盐和干旱胁迫。过表达ThNAC24基因显著降低了刚毛柽柳H_2O_2和超氧阴离子含量,增强了POD和SOD酶的活性,从而减少活性氧(ROS)的积累。过表达ThNAC24基因能够降低刚毛柽柳在逆境胁迫下的电解质渗透率、失水率及MDA的积累,从而保护细胞膜结构的完整性。【结论】刚毛柽柳ThNAC24基因能够响应盐、干旱胁迫,过表达ThNAC24基因植株通过增强POD和SOD活性,进而提高ROS清除能力,减少细胞受损或死亡,从而提高刚毛柽柳的耐盐及抗旱能力。     

Response of clones of Eucalyptus microtheca to NaCl in vitro

We studied the response of nodal segments of Eucalyptus microtheca F.J. Muell. to salt stress in a tissue culture system. Three clones of Eucalyptus microtheca (37, 42 and 43) were grown in vitro under saline conditions (0-140 mM NaCl) for three months. The survival of all three clones decreased with increasing concentrations of NaCl in the medium, but the presence of up to 70 mM NaCl stimulated rooting in Clones 37 and 42. Shoot elongation of Clone 43 was less affected by salt than that of Clones 37 and 42. Leaf growth, expressed as leaf length, was stimulated 40% in Clone 42 and 33% in Clone 43, but was progressively inhibited up to 27% by 70 mM NaCl in Clone 37. Massive accumulation of Na(+) and Cl(-) occurred, especially in Clones 42 and 43 (4000 and 3000 mmol Na(+) kg(DW) (-1), respectively), as the salinity of the culture solution increased, and this was correlated with inhibition of growth. Because of a reduction in the accumulation of K(+) with increasing salinity, the K(+)/Na(+) ratio decreased from a control value of 4.5 to 0.14 in shoots grown in the presence of 140 mM NaCl. The development of techniques for selecting seedlings by monitoring the physiology of shoots in vitro instead of testing whole plants in vivo will provide a relatively simple method of selection for woody trees.     

盐胁迫对尖果沙枣离子分布及渗透调节的影响

为了探讨尖果沙枣的耐盐机理,用NaCl处理尖果沙枣1年生幼苗,处理浓度为50、100、200、300和350 mmol/L,30 d后取样,测定其生理指标。结果表明:随NaCl浓度的增加,叶片膜透性呈增加趋势,叶片水势则有所降低,其中在低于300 mmol/L NaCl浓度下,叶片膜透性和水势的变化幅度不大,在300、350 mmol/L NaCl浓度时与对照呈极显著性差异(P<0.01);随NaCl浓度的增加,脯氨酸含量增加幅度较大,可溶性糖和可溶性蛋白含量均呈较缓的增加趋势,且分别在100 mmol/L和300 mmol/L NaCl浓度下达到最大值,与对照相比,各处理中的有机物质含量均有不同程度增加,其中在350 mmol/L NaCl浓度下的增幅最大;随NaCl浓度的增加,各器官中K 、Ca2 、Mg2 含量变化不大,叶片中K 、Mg2 含量大于茎部和根部,根部Ca2 含量最多,说明植物虽受到一定的伤害,但通过调节渗透调节物质的含量,降低水势,维持其生命代谢,因此,尖果沙枣具有较强的耐盐性。     

Sodium and chloride distribution in salt-stressed Prunus salicina, a deciduous tree species

Measurements were made over four growing seasons of the Na(+) and Cl(-) content of leaves and woody tissues (twigs, branches, trunk and roots) of mature, fruit-bearing Prunus salicina Lindl. (on Marianna 2624 rootstock) trees irrigated during the growing season with water containing 3, 14 or 28 mM salt (2/1 molar ratio of NaCl and CaCl(2)). At the beginning of the study, the trees were 19 years old. Woody tissues of trees irrigated with water containing 14 or 28 mM salt accumulated Na(+) and Cl(-). Leaves of trees irrigated with water containing 14 or 28 mM salt accumulated Cl(-), but not Na(+), unless they had visible symptoms of salt injury. X-Ray microanalysis of leaf mesophyll cells indicated some ability of the cells to sequester Cl(-) in the vacuole. The data demonstrate a capacity for ion compartmentation among tissues and cell organelles in mature Prunus salicina, which may explain the ability of the species to survive low levels of salinity for several years in the field.     

等渗胁迫及其钙调节下青钱柳幼苗叶肉细胞中ATP酶的细胞化学定位及亚细胞变化

研究了等渗的盐和水分胁迫及其钙调节下,青钱柳幼苗叶肉细胞中ATP酶活性在亚细胞中的分布及其超微结构变化。在人工气候室中采用水培法,将青钱柳幼苗进行5个不同浓度处理：对照,85mMNaCl,85mMNaCl＋12mMCa（NO3）2,PEG（渗透势等于85mMNaCl）及PEG（渗透势等于85mMNaCI）＋12mMCa（NO3）2。结果表明：正常生长条件下,ATP酶活性较低并主要定位在细胞核中 等渗胁迫12d以后,ATP酶活性增大并以液泡中居多。等渗水分胁迫下出现的嗜锇颗粒较等渗盐胁迫下的多。由于外源钙的调节作用,等渗胁迫下ATP酶活性增加并主要转移至细胞核上,且在等渗水分胁迫下的嗜锇颗粒明显减少。ATP酶定位在细胞核中表明幼苗遭受胁迫伤害程度较轻,而定位在液泡中则表明受伤害程度较重。等渗处理4和20d后,等渗胁迫及其钙调节下青钱柳幼苗叶片超微结构被破坏程度较无钙调节处理,尤其是较等渗水分胁迫下的轻。初步认为,等渗盐胁迫下青钱柳幼苗遭受的胁迫伤害程度较等渗水分胁迫下的轻,而钙调节作用则以等渗水分胁迫下的效果较佳。     

NaCl胁迫对构树组培苗抗氧化酶活性及同工酶谱的影响(英文)

构树组培苗在50,75,100,125和150 mmol-L-1 NaCl胁迫下,SOD,POD,CAT活性和这3种抗氧化酶同工酶谱的变化情况,以及不同浓度NaCI对可溶性蛋白、脯氨酸和MDA含量的影响.结果表明:NaCI浓度小于100mmol.L-1时可溶蛋白含量升高,随着NaCl浓度的增加可溶蛋白含量降低.低浓度NaCl使SOD活性略有降低,随着浓度升高活性逐渐增强;SOD同工酶谱在NaCI胁迫后变化明显.POD活性变化趋势与SOD相似,而POD同工酶谱略有变化.CAT活性明显提高,而其同工酶谱变化甚微.此外,NaCl胁迫还导致脯氨酸含量明显升高,而MDA含量呈现先升高后降低的趋势.     

Adventitious sprouting of Pinus leiophylla in response to salt stress

Context

Although adventitious shoots are produced in many tree species in response to injury, little is known about the effects of salinity on sprouting.

Aims

The main objective was to examine the sprouting capacity of Pinus leiophylla seedlings in relation to injury and physiological changes induced by NaCl.

Methods

Seedlings were grown in controlled-environment growth rooms and treated with 0, 100, 150, and 200 mM NaCl. Numbers of adventitious shoots were recorded and growth and physiological parameters measured after 64 days of treatments and following a 30-day recovery period.

Results

NaCl treatments triggered sprouting of adventitious shoots, mainly in the lower parts of the stems. However, fewer sprouts were induced by 200 mM NaCl compared with the lower concentration treatments. These changes were accompanied by needle necrosis, decreased chlorophyll concentrations, seedling dry mass, and stem diameter. Stomatal conductance, net photosynthesis, and root hydraulic conductance decreased with increasing NaCl concentrations and did not return to the control levels after 30 days of stress relief.

Conclusions

Pinus leiophylla has the regeneration potential when exposed to salinity. However, very high salt concentrations induce severe physiological impairments and, consequently, a decrease of this regeneration potential.     

Response of Populus x canescens （Populus tremula x alba） to high concentration of NaCl stress

Introduction Salinity is a major factor in inhibiting plant growth and decreasing forest productivity. Up to 1997, the total area affected by salinity in the world had amounted to 930 million hm2 (FAO), and is still increasing. A global study of land use …     

Effect of salt on ROS homeostasis, lipid peroxidation and antioxidant mechanisms in Pinus pinaster suspension cells

? In the Pinus genus, information on the effectiveness of oxidative defence mechanisms during exposure to salt is lacking. The effect of salt stress imposition on ROS homeostasis was investigated using maritime pine (Pinus pinaster Ait.) suspension cells as a model system.

? Cells were maintained in MS-based medium, exposed to salt (50, 100 and 150 mM NaCl) and analysed for biomass production, evidencing a decreasing growth capacity. Use of 100 mM NaCl imposed severe salt stress without affecting cell viability, being chosen for subsequent studies on the ROS homeostasis of salt shock-treated suspension cells.

? Increased total ROS levels were evident on the second day of salt exposure, but a superoxide ion transient burst was immediately noticeable. Additionally, lipid peroxide formation seemed to correlate with superoxide ion breakdown. In-gel superoxide dismutase activity evidenced a FeSOD homodimer with strongly increasing activity between hours 12–48 of salt stress imposition. Subsequently, P. pinaster Fe-Sod1 and csApx1 genes were isolated from a cDNA library and expression was shown to increase within 12–24 h.

? Results show that severe salt treatment generates oxidative stress in P. pinaster cells despite the induction of antioxidant systems, and suggest a putative involvement of ROS in salt stress signalling.

     

Overexpression of mtlD gene in transgenic Populus tomentosa improves salt tolerance through accumulation of mannitol

The mtlD gene encoding mannitol-1-phosphate dehydrogenase, which catalyzes the biosynthesis of mannitol from fructose, was cloned from Escherichia coli and transferred to poplar (Populus tomentosa Carr.) through Agrobacterium-mediated transformation. The transgenic plants were screened and selected on Murashige and Skoog (MS) medium containing 30-50 mg l(-1) kanamycin and verified by polymerase chain reaction (PCR) and Southern blotting. Expression of the gene led to synthesis and accumulation of mannitol in the transgenic plants. Gas chromatography and mass spectrometry (GC/MS) and capillary gas chromatography (GC) showed that transgenic plants accumulated much more mannitol in their tissues than the wild-type plants, whether cultured in vitro, or grown hydroponically or in the field. Increased salt tolerance of transgenic plants was observed both in vitro and in hydroponic culture. The transgenic buds rooted normally on MS medium containing 50 mM NaCl, whereas wild-type buds did not. In the 40-day hydroponic experiments, transgenic poplar plants survived in a 75-mM NaCl treatment, whereas the wild-type poplar plants tolerated only 25 mM NaCl. Under the same NaCl stress, stomatal conductance, transpiration rates and photosynthetic rates were all higher in transgenic plants than in wild-type plants, whereas cellular relative conductivity was lower. We demonstrated that the mtlD gene was expressed in transgenic poplar plants, resulting either directly or indirectly in mannitol accumulation and improved salt tolerance. The constant mannitol concentrations in transgenic plants during the NaCl treatments indicated that mannitol accumulation caused by the mtlD gene was not a consequence of NaCl stress. Height growth was reduced by about 50% in the transgenic plants compared with the wild-type plants in the absence of salt; however, relative growth rate was much less influenced by salt stress in transgenic plants than in wild-type plants. The stunted growth of the transgenic plants may in part explain their improved salt tolerance.     

外源海藻糖对盐胁迫下海滨锦葵种子萌发的影响

以耐盐经济植物海滨锦葵种子为材料,设置浓度为0（CK）、5、10、20、40 g/L NaCl和0（CK）、2、4、8、16 g/L海藻糖为处理液,研究不同浓度的NaCl和海藻糖及二者的交互作用对种子萌发特征的影响。结果表明：NaCl单独处理下,随着盐浓度的提高,显著降低了种子的萌发指标和幼苗鲜重,当盐浓度达到40 g/L时,种子无萌发。对盐处理下的种子施以海藻糖后,低浓度的海藻糖（2 g/L）就可以提高盐胁迫下的种子萌发特性,随着海藻糖浓度的增加,并没有明显提高盐胁迫下的种子萌发。施加一定的外源海藻糖可以提高盐胁迫下幼苗的鲜重,使幼苗更加适应盐胁迫的环境。     

Ectomycorrhizal fungi affect the physiological responses of Picea glauca and Pinus banksiana seedlings exposed to an NaCl gradient

We tested the effects of ectomycorrhizal (ECM) inoculation on greenhouse-grown white spruce (Picea glauca (Moench) Voss) and jack pine (Pinus banksiana L.) seedlings to be used for revegetation of salt-affected tailing sands resulting from the exploitation of oil sand in northeastern Alberta, Canada. White spruce and jack pine seedlings were inoculated with three ECM fungi selected for their in vitro tolerance to excess Na+ and Cl-: Hebeloma crustuliniforme (Bull) Quel. UAMH 5247, Laccaria bicolor Maire (Orton) UAMH 8232 and a Suillus tomentosus (Kauff.) Sing., Snell and Dick isolate from a salt-affected site. The physiological responses of the seedlings to a gradient of NaCl concentration (0, 50, 100 and 200 mM) were assessed over four weeks by: (1) Na+ accumulation and allocation; (2) chlorophyll a fluorescence; (3) growth, (4) water content; and (5) organic osmolyte accumulation. Jack pine seedlings were more sensitive than white spruce seedlings to increasing Na+ and Cl- concentrations. Both species showed decreasing biomass accumulation, and increasing concentrations of organic osmotica and Na with increasing NaCl concentration. White spruce seedlings inoculated with the S. tomentosus isolate had the best growth response at all NaCl concentrations tested. Although jack pine seedlings inoculated with the L. bicolor or S. tomentosus isolate exhibited the highest growth in the 50 and 100 mM NaCl treatments, both fungi increased the photochemical stress and dehydration of their hosts in the 200 mM NaCl treatment. At the latter concentration, jack pine seedlings inoculated with H. crustuliniforme showed the greatest tolerance to salt stress. Although the different fungi altered the physiological response of the host in different ways, inoculation with salt-stress-tolerant ECM fungi increased growth and reduced the negative effects of excess NaCl. Use of controlled mycorrhization may increase survival of coniferous seedlings used for revegetation of salt-affected sites.     

NaCl胁迫下构树幼苗液泡膜生理生化响应(英文)

以构树幼苗根组织和叶组织为试材,测定不同浓度NaCl胁迫下液泡膜H+ -ATPase的活性、液泡膜脂肪酸组成和膜的流动性.结果表明:在低浓度NaCl胁迫下,根组织中液泡膜H+ -ATPase的活性有所增加,当胁迫浓度为150 mmol·L-1时,活性又有所下降,但接近对照水平.而叶组织液泡膜H+ -ATPase的活性在100 mmol·L-1时降低,高浓度NaCl(150 mmol·L-1)处理后又有所升高.液泡膜脂肪酸基本组成为C16:0,C16:1,C18:0,C18:1,C18:2和C20:0.盐胁迫后构树幼苗液泡膜脂肪酸组分相对含量发生变化.根组织液泡膜脂肪酸不饱和度下降,150 mmol·L-1 NaCl处理后有所升高;而在叶组织中,液泡膜脂肪酸不饱和度在50 mmol·L-1 NaCl处理后下降,随着盐胁迫浓度的增加,不饱和度又高于对照.NaCl胁迫下,构树液泡膜流动性和脂肪酸不饱和度的变化趋势相一致.脂肪酸不饱和度下降时,膜流动性下降,反之则膜流动性上升.研究结果为深入了解木本植物液泡膜H+ -ATPase活性、脂肪酸组成和膜的流动性在盐胁迫下的适应及其3者的相互关系提供了参考.     

Sodium chloride improves photosynthesis and water status in the succulent xerophyte Zygophyllum xanthoxylum

Zygophyllum xanthoxylum, a C(3) woody species, is a succulent xerophyte that is well adapted to arid environments. Our previous investigations showed that Na(+) has a positive effect on the growth of Z. xanthoxylum under drought conditions, which was closely related to high Na(+) accumulation in leaves. To reveal the physiological mechanisms underlying how Na(+) accumulation improves the drought resistance of Z. xanthoxylum, 3-week-old seedlings were treated with a series of additional external NaCl concentrations (5-150 mM) in sand culture experiments. Seedlings were also subjected to water deficit (30% of field water capacity) in the presence or absence of additional NaCl (50 mM) in pot experiments. The results indicated that 50 mM NaCl could mitigate deleterious impacts of water deficit on the growth of Z. xanthoxylum, by improving the relative water content, inducing a significant drop in leaf water potential and, concomitantly, increasing leaf turgor pressure and chlorophyll concentrations resulting in an enhancement of overall plant photosynthetic activity (i.e., photosynthetic rate and water use efficiency). Furthermore, NaCl (50 mM) could alleviate the inhibitory effect of water deficit on the activity of photosystem II in Z. xanthoxylum. The contribution of Na(+) to the total osmotic potential varied from 8% in the control to 13% in plants subjected to water deficit and, surprisingly, to 28% in plants grown in the presence of 50 mM NaCl under water deficit; however, the contribution of K(+) significantly decreased from 13 to 8%. These findings suggest that, under arid environments, Z.?xanthoxylum is able to accumulate a high concentration of Na(+) in its leaves and use it directly for osmotic adjustment, which was coupled with an improvement in leaf hydration and photosynthetic activity.     

Gas exchange, water relations and osmotic adjustment in Phillyrea latifolia grown at various salinity concentrations

Leaf gas exchange, water relations and osmotic adjustment were studied in hydroponically grown Phillyrea latifolia L. plants exposed to 5 weeks of salinity stress (0, 80, 160, 240 and 320 mM NaCl) followed by 5 weeks of treatment with half-strength Hoagland solution. Whole-plant relative growth rate and root/shoot and lateral/structural root ratios were also evaluated. Net CO2 assimilation rate, stomatal conductance and transpiration rate were markedly decreased by all of the salt treatments. Growth was also strongly depressed by all salt treatments, especially lateral root growth. Leaf water potential decreased soon after salinity stress was imposed, whereas there was a lag of several weeks before leaf osmotic potential decreased in response to the salt treatments. After 5 weeks of salinization, leaf turgor of salt-treated plants was similar to that of controls. Although Na+ + Cl- contributed little to the salt-induced changes in osmotic potential at full turgor (Psi(piFT)), the contributions of K+, mannitol (Man) and glucose (Glc) to Psi(piFT) markedly increased as external salinity increased. Salt accumulation was negligible in the youngest leaves, which mostly accumulated soluble carbohydrates and K+; in contrast, old leaves served as storage sinks for Na+ and Cl-. Photosynthetic performance of salt-treated plants fully recovered once salt was leached from the root zone, with the recovery rate depending on the severity of the salt stress previously experienced by the plants. Recovery of gas exchange occurred even though the leaves still had a salt load similar to that detected in leaves at the end of the 5-week salinity period, and had markedly lower concentrations of K+ and soluble carbohydrates than control leaves. We conclude that salt-induced water stress primarily controlled gas exchange of salt-treated P. latifolia leaves, whereas the salt load in the leaves did not cause irreversible damage to the photosynthetic apparatus.     

盐碱胁迫对2种地被竹叶绿素含量的影响

以黄条金刚竹(Sasaella masamuneana f. aureostriata)、铺地竹(Pleioblastus argenteostriatus)为试材,将2种中性盐NaCl、Na₂SO₄和2种碱性盐 NaHCO₃、Na₂CO₃按不同比例混和,模拟出盐碱度各不相同的盐碱条件,研究不同盐碱胁迫处理对2种地被竹叶绿素含量的影响,旨在为今后优良耐盐地被竹选择及有效应用提供参考。结果表明,低浓度中性盐胁迫下,黄条金刚竹叶绿素a含量呈现“升高—降低—升高”的趋势,其余处理叶绿素a含量变化较为平缓,说明植株始终受到抑制;铺地竹受到胁迫后,叶绿素a在胁迫初期均呈现上升趋势,但随着胁迫时间的推移,叶绿素a含量呈现明显的下降趋势,尤其以盐浓度300 mmol/L处理最为明显。2种竹叶绿素b含量变化基本呈现初期上升后期下降的趋势。碱性盐胁迫下,2种竹的叶绿素a和b含量变化规律不明显,但是含量总体呈现下降的趋势。     

Effects of salinity and desalination on seed germination of six annual weed species

The effects of various salinities and desalination on seed germination of six annual glycophytes (Artemisia sieversiana, A. scoparia, Chloris virgata, Eragrostis pilosa, Chenopodium acuminatum and Chenopodium glaucum) were studied in Horqin Sandy Land, Inner Mongolia, China. NaCl solutions of five concentrations (0 mM, as the control, and 50, 100, 200 and 300 mM) were used for saline stress and desalination treatments. Increasing salinity significantly reduced germination percentages of A. sieversiana, A. scoparia, Ch.virgata and Ch. acuminatum, but had no effect on the germination percentages of E. pilosa. Lower salinity levels (50 mM) significantly increased germination percentage of Ch. glaucum. High salinity might be a precondition for germi- nation after desalination for five of the six species, excepting E. pilosa at NaCl concentration of 300 mM in comparison with non-primed seeds. Higher salinity (>200 mM) led to some specific ion toxicity and reduced seed viability of A. sieversiana. No specific ion toxicity but an osmotic effect limited the germination of other five species was observed The final germination percentages (salinity stress and desalination) of the six species showed three variations in comparison with the controls, namely, indiscrimination, stimulation, and reduction. Germination responses to salinity and desalination suggested that the six species were separated into three categories. Three species (A. sieversiana, Ch. virgata and Ch. acuminatum) showed similar germination responses to salinity with those of halophyte, but also showed a lower tolerance limit than most halophytes, although this was not always the case. A. scoparia and Ch. glaucum exhibited some ’salt stimulation’ in seed germination percentages after desalination, whereas E. pilosa did not show any obvious responseto salinity. Therefore, salinity usually induces dormancy of seeds with strong germination capacity in fresh water, but has few, or even positive, effects on seeds with strong innate dormancy.