Overexpression of AtNHX5 improves tolerance to both salt and drought stress in Broussonetia papyrifera (L.) Vent

Paper mulberry (Broussonetia papyrifera L. Vent) is well known for its bark fibers, which are used for making paper, cloth, rope, etc. It was found that, in addition to its well-documented role in the enhancement of plant salt tolerance, overexpression of the Na+/H+ antiporter (AtNHX5) gene in paper mulberry plants showed high drought tolerance. After exposure to water deficiency and salt stress, the wild-type (WT) plants all died, while the AtNHX5-overexpressing plants remained alive under high salt stress, and had a higher survival rate (>66%) under drought stress. Measurements of ion levels indicated that Na+ and K+ contents were all higher in AtNHX5-overexpressing leaves than in WT leaves in high saline conditions. The AtNHX5 plants had higher leaf water content and leaf chlorophyll contents, accumulated more proline and soluble sugars, and had less membrane damage than the WT plants under water deficiency and high saline conditions. Taken together, the results indicate that the AtNHX5 gene could enhance the tolerance of paper mulberry plants to multiple environmental stresses by promoting the accumulation of more effective osmolytes (ions, soluble sugars, proline) to counter the osmotic stress caused by abiotic factors.     

Overexpression of the ThTPS gene enhanced salt and osmotic stress tolerance in Tamarix hispida

Trehalose is a non-reducing disaccharide with high stability and strong water absorption properties that can improve the resistance of organisms to various abi-otic stresses.Trehalose-6-phosphate synthase (TPS) plays important roles in trehalose metabolism and signaling.In this study,the full-length cDNA of ThTPS was cloned from Tamarix hispida Willd.A phylogenetic tree includ-ing ThTPS and 11 AtTPS genes from Arabidopsis indicated that the ThTPS protein had a close evolutionary relationship with AtTPS7.However,the function of AtTPS7 has not been determined.To analyze the abiotic stress tolerance function of ThTPS,the expression of ThTPS in T.hispida under salt and drought stress and JA,ABA and GA3 hormone stimu-lation was monitored by qRT-PCR.The results show that ThTPS expression was clearly induced by all five of these treatments at one or more times,and salt stress caused par-ticularly strong induction of ThTPS in the roots of T.hispida.The ThTPS gene was transiently overexpressed in T.his-pida.Both physiological indexes and staining results showed that ThTPS gene overexpression increased salt and osmotic stress tolerance in T.hispida.Overall,the ThTPS gene can respond to abiotic stresses such as salt and drought,and its overexpression can significantly improve salt and osmotic tolerance.These findings establish a foundation to better understand the responses of TPS genes to abiotic stress in plants.     

Over expression of TaFer gene from Tamarix androssowii improves iron and drought tolerance in transgenic Populus tomentosa

Ferritin, a universal intracellular protein, can store large amounts of iron and improve plant resistance to abiotic and biotic stress. In this study, a ferritin gene(TaFer) from Tamarix androssowii Litv. was transferred into Populus tomentosa Carr. cv 'BJR01' via Agrobacterium. Six independent transgenic lines were obtained with a tolerance to kanamycin and three were randomly selected for further analysis. The PCR and RT-PCR results indicate that the TaFer gene had been integrated into the poplar genome. The effect of the gene on abiotic stress tolerance was tested, and the results show that transgenic plants improve growth, had higher chlorophyll and lower MDA contents, and higher relative electrical conductivity,fewer changes of SOD and POD activities, higher iron content, higher root ferric reductase activity and lower levels of ROS accumulation and cell death in response to drought, Fe-insufficient or Fe-excess tolerance. These results indicate that the TaFer gene can improve abiotic stress tolerance in transgenic Populus tomentosa.     

Effects of water stress on growth,dry matter allocation and water-use efficiency of a leguminous species, <Emphasis Type="Italic">Sophora davidii</Emphasis>

The adaptation responses to different water conditions and the drought tolerance of Sophora davidii seedlings were assessed in a greenhouse experiment. Two-month-old seedlings were subjected to the following water supplies for 95 days: 100, 80, 60, 40 and 20% of field water capacity. The seedlings at 100% FC had the greatest productivity, height, basal diameter, branch number, leaf number and leaf area. Water supply <80% FC was the threshold of drought-initiated negative effects on seedling growth, yield and physiological processes; these parameters were severely reduced at 20% FC, however, there was no plant death during the experiment. Moreover, water stress decreased leaf relative water content, specific leaf area, leaf area ratio, and water-use efficiency (WUE), whereas it increased the biomass allocation to roots, which resulted in a higher root:stem mass ratio under drought. The S. davidii seedlings tolerated drought by maintaining high leaf relative water content and by reducing branching and leaf expansion. However, low productivity and WUE at 20% FC suggested that seedlings did not produce high biomass under severe drought. Therefore, prior to introducing S. davidii in forestation efforts, a water supply >40% FC is recommended for seedlings to maintain growth and productivity. These results provide insights into limitations and opportunities for establishment of S. davidii in arid regions.     

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。     

Cloning of four DREB genes from Tibetan Sophora moorcroftiana and analysis of their expression during abiotic stress

Sophora moorcroftiana is an endemic, droughtresistant shrub that grows in Tibet and has some degree of resistance to salt, cold, heat, and drought. In the present study, four dehydration responsive element-binding(DREB) genes(Sm DREB1, Sm DREB2, Sm DREB and Sm DREB1) were isolated from S. moorcroftiana for the first time and their expression and proline content under abiotic stress were analyzed. Proline accumulated in seedlings under drought, salt, cold, and heat stress treatments. The four genes were variously expressed in response to the four abiotic stresses. Sm DREB1 was induced by drought, cold, and heat stresses; Sm DREB2 and Sm DREB4 were both induced by salt, cold, and heat stresses, whereas Sm DREB3 was induced by drought and heat stresses. Thus, these four genes may participate in conferring tolerance to these four abiotic stresses and are candidate genes for genetic engineering in the future.     

Verification of the resistance of a LEA gene from Tamarix expression in Saccharomyces cerevisiae to abiotic stresses

The role of late embryogenesis abundant （LEA） proteins in stress tolerance was examined by using a yeast expression system. LEA protein tolerance to the abotic stresses in plants involved in salt, drought and freezing stresses and additional tolerance to heat, NaHCO3 （salt-alkali） and ultraviolet radiation was also investigated. The transgenic yeast harboring the Tamarix LEA gene （DQ663481） was generated under the control of inducible GAL promoter （pYES2 vector）, yeast cells transformed with pYES2 empty vector were also generated as a control. Stress tolerance tests showed that LEA yeast transformants exhibited a higher survival rates than the control transformants under high temperature, NaHCO3, ultraviolet radiation, salt （NaCl）, drought and freezing, indicating that the LEA gene is tolerant to these abiotic stresses. These results suggest that the LEA gene is resistant to a wider repertoire of stresses and may play a common role in plant acclimation to the examined stress conditions.     

Two novel eukaryotic translation initiation factor 5A genes from Populus simonii × P. nigra confer tolerance to abiotic stresses in Saccharomyces cerevisiae

The role of plant e IF5 A proteins in multiple biological processes, such as protein synthesis regulation,translation elongation, m RNA turnover, programmed cell death and stress tolerance is well known. Toward using these powerful proteins to increase stress tolerance in agricultural plants, in the present study, we cloned and characterized Psne IF5A2 and Psne IF5A4 from young poplar(P. simonii 9 P. nigra) leaves. The deduced amino acid sequences of Psne IF5A2 and Psne IF5A4 were 98 %similar to each other, and they are orthologs of e IF5A1 in Arabidopsis. In a subcellular localization analysis,Psne IF5A2 and Psne IF5A4 proteins were localized in the nucleus and cytoplasm. q RT-PCR analysis showed that Psne IF5A2 and Psne IF5A4 were transcribed in poplar flowers, stem, leaves, and roots. In addition, they were also induced by abiotic stresses. Transgenic yeast expressing Psne IF5A2 and Psne IF5A4 had increased salt, heavy metal, osmotic, oxidative tolerance. Our results suggest that Psne IF5A2 and Psne IF5A4 are excellent candidates for genetic engineering to improve salt and heavy metal tolerance in agricultural plants.     

Enhanced tolerance of photosynthesis to high-light and drought stress in Pseudotsuga menziesii seedlings grown in ultraviolet-B radiation

We investigated the effects of an ambient dose of ultraviolet-B (UV-B) radiation on chamber-grown Pseudotsuga menziesii var. glauca (Beissn.) Franco (Douglas-fir) seedlings, to determine if the presence of UV-B radiation in the growth light regime induces tolerance to environmental stresses such as high light and drought. Douglas-fir seedlings were grown without UV-B radiation or with 6 kJ m-2 day-1 of biologically effective UV-B, which is ambient for the intermountain regions of Idaho. Non-stressed seedlings grown with UV-B radiation had 35% lower seedling dry mass, 36% higher concentrations of UV-B absorbing compounds per unit leaf area, 30% lower stomatal frequencies, 25% lower light-saturated photochemical efficiencies of Photosystem II and 45% lower light-saturated stomatal conductance than non-stressed seedlings grown without UV-B radiation. After 4 days of high-light stress, seedlings grown with UV-B radiation had 32% higher light-saturated carbon assimilation rates (A(CO2)) than seedlings grown without UV-B radiation. After water was withheld from the seedlings for up to 15 days, seedlings grown with UV-B radiation had 50% higher A(CO2) and 40% higher seedling water potentials than seedlings grown without UV-B radiation. The results support the hypothesis that UV-B radiation can act as an environmental signal to induce tolerance to high-light and drought stress in Douglas-fir seedlings. Possible mechanisms for the enhanced stress tolerance are discussed.     

盐渍化土壤改良利用新方法——植物耐盐基因的遗传转化

土壤盐碱化是目前全球最严重的环境问题之一,包括人口膨胀在内的各方面因素使人类不断开发和利用大面积的土地,逐渐生成新的次生盐渍化,加速了土壤盐碱化的进程。通过农业生物技术培育耐盐植物品种或开发利用有经济价值的盐生植物资源以改良土壤已成为研究热点。文中综述了植物耐(抗)盐碱逆境胁迫的相关基因和外源基因遗传转化的方法,并对现今生物耐(抗)盐碱逆境胁迫的基因转化研究进行了展望。     

Expression of a carrot 36 kD antifreeze protein gene improves cold stress tolerance in transgenic tobacco

Antifreeze proteins （AFPs） enable organisms to survive under cold conditions, and have great potential in improving cold tolerance of cold-sensitive plants, In order to determine whether expression of the carrot 36 kD antifreeze protein gene confers improved cold-resistant properties to plant tissues, we tried to obtain transgenic tobacco plants which expressed the antifreeze protein. Cold, salt, and drought induced promoter Prd29A was cloned using PCR from Arabidopsis. Two plant expression vectors based on pBI121 were constructed with CaMV35S：AFP and Prd29A：AFP. Tobacco plantlets were transformed by Agrobacterium-medicated transformation. PCR and Southern blotting demonstrated that the carrot 36 kD afp gene was successfully integrated into the genomes of transformed plantlets. The expression of the afp gene in transgenic plants led to improved tolerance to cold stress. However, the use of the strong constitutive 35S cauliflower mosaic virus （CaMV） promoter to drive expression of afp also resulted in growth retardation under normal growing conditions. In contrast, the expression of afp driven by the stress-inducible Prd29A promoter from Arabidopsis gave rise to minimal effects on plant growth while providing an increased tolerance to cold stress condition （2℃）. The results demonstrated the prospect of using Prd29A-AFP transgenic plants in cold-stressed conditions that will in turn benefit agriculture.     

Regeneration and gene transformation systems of Robinia pseudoacacia ''Idaho'' mediated by Agrobacterium tumefaciens

Robinia pseudoacacia 'Idaho' is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentration of it was 8 mg·L-1.     

刺槐和紫穗槐抗旱性比较试验

通过采取不同时间的水分控制处理对刺槐（Robinia pseudoacacia）和紫穗槐（Amorpha fruticosa）2树种的抗旱能力进行了试验比较,结果表明：当受到水分胁迫时,2种植物体内Pro含量均有不同程度的升高,紫穗槐Pro含量的峰值为184.37μg·g-1,刺槐的峰值为119.35μg·g-1,刺槐的耐脱水能力比紫穗槐弱;从干旱胁迫条件下2植物体内超氧化物歧化酶活性、丙二醛含量、相对含水量和水分饱和亏缺等指标的变化也得出紫穗槐的抗旱性强于刺槐。     

干旱胁迫对麻栎无性系蒸腾作用的影响

以13个3年生麻栎无性系为研究对象,采用盆栽试验方法,研究了在持续干旱胁迫条件下的麻栎无性系土壤水势、叶片水势光合速率和蒸腾速率变化规律及其关系。结果表明,随着干旱胁迫的加剧,13个麻栎无性系土壤水势呈现出不断下降的变化趋势,叶片水势的变化无一致规律,光合速率则先下降而后上升,蒸腾速率的变化规律不尽相同。在13个麻栎无性系中,2、4、5、7、9和16号无性系的蒸腾速率、叶片水势和光合速率具有相同规律,随着干旱胁迫的加剧先下降而后上升,具有较强的适应干旱逆境的能力。对不同干旱条件下无性系的蒸腾速率、土壤水势、叶片水势和光合速率的相关性分析发现,干旱胁迫7天时,蒸腾速率和光合速率达极显著正相关;干旱胁迫12天时,蒸腾速率和土壤水势间呈极显著负相关,光合速率与土壤水势呈显著负相关;干旱胁迫17天时,蒸腾速率与光合速率呈显著正相关。     

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。     

Seasonal changes in photosynthesis of trees in the flooded forest of the Mapire River

We studied the flood tolerance of five tree species growing in the flooded forest adjacent to the Mapire river, in SW Venezuela. Mean photosynthetic rate and leaf conductance were 11 &mgr;mol m(-2) s(-1) and 700 mmol m(-2) s(-1), respectively. Xylem water potential ranged from -0.08 to -1.15 MPa. Based on leaf gas exchange as a criterion of tolerance to flooding, two response patterns were identified: (1) decreasing photosynthetic rate with increasing flooding and leaf conductance (Psidium ovatifolium Berg. ex Desc., Campsiandra laurifolia Benth., Symmeria paniculata Benth. and Acosmium nitens (Vog.) Benth); and (2) independence of photosynthesis and leaf conductance from flooding (Eschweilera tenuifolia (Berg.) Miers.). In the first response pattern, declining photosynthetic rate with flooding may be interpreted as a sign of reduced flood tolerance, whereas the second response pattern may indicate increased flood tolerance. An increase in xylem water potential with depth of water column was found for all species (with the possible exception of P. ovatifolium), indicating that flooding does not cause water stress in these trees. Submerged leaves that had been under water for between four days and four months generally had photosynthetic rates and leaf conductances similar to those of aerial leaves, indicating maintenance of photosynthetic capacity under water. Daily positive oscillations in glucan content in submerged leaves of P. ovatifolium and C. laurifolia suggest that submerged leaves do not represent a sink for photosynthates produced by aerial leaves.     

Growth,biomass partitioning,and water-use efficiency of a leguminous shrub (<Emphasis Type="Italic">Bauhinia faberi</Emphasis> var. <Emphasis Type="Italic">microphylla</Emphasis>) in response to various water availabilities

Responses of the endemic leguminous shrub Bauhinia faberi var. microphylla, to various soil water supply regimes were studied in order to assess water stress tolerance of seedlings. Two-month-old seedlings were grown under water supply regimes of 100, 80, 60, 40, and 20% water field capacity (FC), respectively, in a temperature and light-controlled greenhouse. Plant height and leaf number were measured monthly over a 4-month period, while water use (WU), water-use efficiency (WUE), leaf relative water content (RWC), biomass production and its partitioning were recorded at the end of the experiment. Seedlings exhibited the greatest biomass production, height, basal diameter, branch number, leaf number, and leaf area when soil content was at 100% FC, and slightly declined at 80% FC. These parameters declined significantly under 60% FC water supply, and severely reduced under 40 and 20% FC. RWC, WU and WUE decreased, while the ratio of root mass to stem mass (R:S) increased in response to decreasing water supply. Water stress caused leaf shedding, but not plant death. The results demonstrated that B. faberi var. microphylla seedlings could tolerate drought by reducing branching and leaf area while maintaining a high R:S ratio. However, low dry mass and WUE at 40 and 20% FC suggested that the seedlings did not produce significant biomass under prolonged severe water deficit. Therefore, before introducing B. faberi var. microphylla in vegetation restoration efforts, water supply above 40% FC is recommended for seedlings to maintain growth.