Identification of microRNAs and their targets from Populus euphratica

Populus euphratica is an ideal model system for research into the abiotic stress resistance research of woody plants. Although microRNAs have been found in poplars and have been shown to have diverse biological functions, a majority of them are genus- or specie-specific and few microRNAs have been found in P. euphratica to date. In this study, microRNA cloning and computational expressed sequence tag analysis were used to identify 72 putative miRNA sequences in P. euphratica. These sequences could be classified into 21 families, 12 of which were novel, increasing the number of known poplar microRNA families from 42 to 54. Expression analysis indicated that five P. euphratica microRNAs were induced by dehydration stress. Bioinformatics prediction showed that the 130 target genes are involved in development, resistance to stress, and other cellular processes. These results suggest several roles for miRNAs in the regulatory networks associated with the abiotic stress resistance of tree species.     

基于遥感图像处理技术胡杨叶气孔密度的估算及其生态意义

拟利用遥感图像处理技术--面向对象分类,计算胡杨叶片气孔密度,采用面向对象分类的专业软件eCognition对气孔图像进行多尺度分割,将生成的分类图像导入ArcGIS中计算气孔密度,最后用R语言编写代码进行批处理。研究结果显示:该方法用于计算叶片气孔的密度精度高;18个样点胡杨气孔密度存在着较大的差异,从76.7 个/mm²到139.4 个/mm²不等,其平均密度为105 个/mm²;随着干旱胁迫加强,气孔密度表现下降上升再下降的趋势。     

塔里木河下游地下水埋深对胡杨气体交换和叶绿素荧光的影响

为了分析干旱环境下地下水埋深变化对胡杨(Populus euphratica oliv.)光合作用的影响,对塔里木河下游3个地下水埋深(4.91,6.93m和8.44m)环境下胡杨叶片的气体交换日变化、光响应曲线、PN-C_i曲线以及叶绿素荧光特性等进行了比较研究。研究结果表明:当地下水埋深从4.91m增加到6.93m和8.44m,胡杨光合速率(P_N)(10:00),初始荧光(F₀)、最大荧光(F_m)、以及PSⅡ实际光化学效率(Ω_PSⅡ)、电子传递速率(ETR)、非光化学猝灭系数(NPQ)和正午叶水势(Ψ_midday)等都发生了明显变化,其中胡杨NPQ增加了109% 127%,Ω_PSⅡ,ETR和Ψ_midday分别减小了24% 29%,17% 22%和31.6% 45.6%,表明胡杨受到的干旱胁迫程度在增加;而当地下水埋深在6.93 8.44m之间时,上述参数无显著变化,表明胡杨很可能处于相同干旱胁迫程度;并且在地下水埋深4.91 8.44m范围内,最大光化学效率(F_v/F_m),表观量子效率(φ),Rubisco羧化速率(V_cmax), 等参数都未发生明显变化,表明即使地下水埋深增加到8.44m,此时的干旱胁迫程度也未超过胡杨的耐受能力,其光合能力也未受到不可逆转的伤害。     

不同林龄胡杨克隆繁殖根系分布特征及其构型

以中龄林和成熟林胡杨为研究对象,采用挖剖面和根窗的方法,研究胡杨繁殖根系分布、根系构型,以及胡杨根蘖与繁殖根系构型之间的关系。结果表明:(1)细根(d<2 mm)的根长密度、根表面积密度,随深度增加呈现指数函数分布;(2)中龄林细根的根长密度、根表面积密度在0—90 cm各层都是显著大于成熟林的对应指标(P<0.05),成熟林的中等粗根(5 mm0.05),且两种林龄的一级侧根数、分枝角度亦无显著差异(P>0.05);(5)对比两种林龄不同根序上的根蘖芽发现,二级根上不定芽个数均是同组一级根上不定芽个数的3—4倍;基于以上对胡杨根系的功能权衡的分析,得出:细根对胡杨根系构型有重要的影响,在胡杨根系功能权衡中扮演重要角色。     

Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants

A rice gene encoding a calcium-dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over-expression of OsCDPK7 enhanced induction of some stress-responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post-translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement.     

Over-expression of calcium-dependent protein kinase 13 and calreticulin interacting protein 1 confers cold tolerance on rice plants

Calcium is a ubiquitous signaling molecule and changes in cytosolic calcium concentration are involved in plant responses to various stimuli. The rice calcium-dependent protein kinase 13 (CDPK13) and calreticulin interacting protein 1 (CRTintP1) have previously been reported to be involved in cold stress response in rice. In this study, rice lines transformed with sense CDPK13 or CRTintP1 constructs were produced and used to investigate the function of these proteins. When the plants were incubated at 5°C for 3 days, leaf blades of both the sense transgenic and vector control rice plants became wilted and curled. When the plants were transferred back to non-stress conditions after cold treatment, the leaf blades died, but the sheaths remained green in the sense transgenic rice plants. Expression of CDPK13 or CRTintP1 was further examined in several rice varieties including cold-tolerant rice varieties. Accumulation of these proteins in the cold-tolerant rice variety was higher than that in rice varieties that are intermediate in their cold tolerance. To examine whether over-expression of CDPK13 and CRTintP1 would have any effect on the proteins or not, sense transgenic rice plants were analyzed using proteomics. The 2D-PAGE profiles of proteins from the vector control were compared with those of the sense transgenic rice plants. Two of the proteins that differed between these lines were calreticulins. The results suggest that CDPK13, calreticulin and CRTintP1 might be important signaling components for response to cold stress in rice.     

干旱胁迫下胡杨光合光响应过程模拟与模型比较

以塔里木干旱荒漠区2年生胡杨幼苗为试材,盆栽模拟荒漠生境5种水分梯度,利用Li-6400便携式光合作用系统测定胡杨在干旱胁迫下光合作用的光响应过程,并采用4种光响应模型对其进行拟合与比较,以期优选出适用于干旱荒漠环境的光响应模型,阐明胡杨光合作用对干旱胁迫的响应规律与适应机制。结果表明:胡杨净光合速率(P_n)随干旱胁迫加剧呈下降趋势,同一光强(PAR)下P_n降幅增大。中度干旱胁迫以下(土壤相对含水量,RSWC45%)胡杨在高PAR下仍能维持相对较高P_n,光抑制程度轻;直角双曲线、非直角双曲线和指数模型均可较好地模拟P_n-PAR响应过程,但最大净光合速率(P_(nmax))、光饱和点(LSP)拟合值与实测值差异极显著(P0.01)。中度干旱胁迫以上(RSWC45%)胡杨P_n随PAR升高而显著下降,LSP与P_(nmax)极显著降低,光抑制现象明显;仅直角双曲线修正模型拟合的胡杨光响应过程、光响应参数与实际情况较吻合。4种模型模拟效果顺序:直角双曲线修正模型指数模型非直角双曲线模型直角双曲线模型。4种光响应模型对干旱胁迫具有不同的适应性,直角双曲线修正模型适用于各种水分条件,尤其适用于干旱荒漠生境,其它3种模型适用于水分条件较好的生境。光响应特征参数对干旱胁迫的响应阈值不同。随干旱胁迫加剧,胡杨表观量子效率(AQY)、P_n、LSP与P_(nmax)持续降低,严重干旱胁迫下暗呼吸速率(R_d)、LCP反而明显增大。RSWC45%胡杨仍能保持较高的AQY、P_(nmax)、LSP,RSWC45%其P_(nmax)、LSP显著降低,干旱胁迫显著抑制了胡杨光合进程和光强耐受范围,降低了光合效率,严重干旱胁迫严重影响胡杨苗木的正常生长和光合作用。干旱荒漠环境下,胡杨采取缩窄光照生态幅、降低光能利用率和减少呼吸消耗来积极抵御荒漠干旱逆境伤害的生态对策。因此,从极端干旱荒漠区种群保护与植被恢复角度来看,胡杨林土壤水分应维持在RSWC 50%左右,符合干旱缺水地区植物生长和高效用水的管理原则。     

干旱胁迫下胡杨实生幼苗氮素吸收分配与利用

胡杨(Populus euphratica)是塔里木河流域荒漠河岸林的建群种,水分和氮素是限制胡杨幼苗的存活及早期生长的主要因子。利用~(15)N同位素示踪技术分析水和氮素的交互作用对胡杨幼苗不同生长阶段氮素的吸收分配利用及幼苗生长的影响,进一步探究氮素对胡杨实生苗早期形态建成的作用及对干旱胁迫的缓解效应,以期提高幼苗的存活率。实验以一年生胡杨实生幼苗为研究对象,采用温室内盆栽实验,设置4个干旱处理(D_1、D_2、D_3、D_4,土壤相对含水量为:20%—25%、40%—45%、60%—65%、80%—85%)和3种氮素水平(N_0、N_1、N_2:0、3、6 g/盆)测定胡杨幼苗的生长指标和各部分的Ndff、分配率及利用率。结果表明:胡杨幼苗在土壤相对含水量60%—65%(D_3)、氮素添加量3 g/盆(N_1)时,其生长表现为最佳状态;干旱胁迫下,不同氮素添加量对胡杨幼苗各部分的Ndff值存在显著差异,N_2低于N_1;随干旱胁迫减弱(D_3、D_4),植株在生长早期(25 d)根部吸收的~(15)N优先向地上部分转运,生长后期(75 d)植株Ndff最高,其中以根系中Ndff最高;不同生长期幼苗各部分的~(15)N分配存在显著差异,根系~(15)N分配率较高,但不同氮量处理间差异不显著;随生长期的推移,植株对~(15)NH_4~(15)NO_3的利用率表现为粗根最大,各处理中D_3N_1处理均显著高于其他处理。结论:轻度干旱胁迫下添加适量的氮素能够增强植株对氮素的吸收征调能力,优化水资源获取以维持生存的重要机制。     

塔里木河上游胡杨种群结构与动态

采用样带调查法对塔里木河上游3个断面8.5 hm2天然胡杨林进行野外调查,绘制了胡杨种群年龄结构图和存活曲线,结合种群动态量化方法与时间序列预测,分析种群结构与动态特征。结果表明:塔里木河上游胡杨种群密度随径级增大而减小,自疏指数接近-3/2,个体胸径达24 cm后种群密度保持相对稳定。中、下段胡杨种群年龄结构呈金字塔型,幼龄级所占比例较大、林分年轻;存活曲线为Deevey C型,且动态指数0,为增长种群。受所处生境条件影响,上段不同生境胡杨种群年龄结构差异明显。阿瓦提县胡杨种群结构呈金字塔型,幼龄级比例高达77.2%,存活曲线属Deevey C型,为增长种群;南口镇与托海乡胡杨种群结构呈壶型,幼龄级比例极低、中大龄级比例高达84.7%,存活曲线属Deevey A型,为衰退种群;沙黑里克镇胡杨种群结构呈钟型,存活曲线属Deevey A型,为暂时稳定种群。种群数量动态与时间序列分析表明,中、下段胡杨更新资源丰富,未来种群能够稳定增长;上段南口镇、托海乡与沙黑里克镇幼龄级数量随时间推移减少,种群向大老龄级发展,未来种群将走向衰退。总之,上段胡杨种群因更新资源不足而趋于衰退,未来保持适当的人工辅助恢复措施和加强保护现有植株及其生境是保持胡杨种群自然更新和进行种群恢复的关键。     

Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing

Salt is one of the main environmental factors limiting plant growth and a better understanding of mechanisms of salt stress would aid efforts to bolster plant salt tolerance. MicroRNAs are well known for their important regulatory roles in response to abiotic stress in plants. In this study, high-throughput sequencing was employed to identify miRNAs in Populus tomentosa plantlets treated or not with salt (200 mM for 10 h). We found 141 conserved miRNAs belonging to 31 families, 29 non-conserved but previously-known miRNAs belonging to 26 families, and 17 novel miRNAs. Under salt stress, 19 miRNAs belonging to seven conserved miRNA families were significantly downregulated, and two miRNAs belonging to two conserved miRNA families were upregulated. Of seven non-conserved miRNAs with significantly altered expression, five were downregulated and two were upregulated. Furthermore, eight miRNAs were validated by qRT-PCR and their dynamic differential expressions were analyzed. In addition, 269 target genes of identified miRNAs were predicted and categorized by function. These results provide new insights into salt-responsive miRNAs in Populus.     

黑河下游绿洲胡杨生长状况与叶生态特征

选择黑河下游额济纳绿洲为研究区,以优势物种胡杨(Populus euphratica Oliv.)为研究对象。在2009年和2010年对研究区胡杨的生长状况、叶生态特征和生境进行了调查。调查结果表明:胡杨受水分胁迫的程度增强,生长态势变差,其枯枝比由2.45%增加到81.00%,其比叶面积由11.84 m2/kg减少到5.35 m2/kg。叶气孔密度变化很大,最小值为105(个/mm2),最大值为218(个/mm2),平均值为158.4(个/mm2),随着地下水埋深的增加,叶气孔密度先减少后增加之后再显著减少(P0.05),呈三次函数关系。研究得出枯枝比能够反映胡杨分布区地下水的变化状况,是林相描述比较重要的指标,比叶面积和叶气孔密度能够指示胡杨种群环境变化。研究结果可为荒漠河岸林恢复和生态输水效应评价提供科学依据。     

Effects of endophyte infection on drought stress tolerance of Lolium perenne accessions from the Mediterranean region

Perennial ryegrass (Lolium perenne) is a cool-season grass species that is often infected by the leaf-inhabiting endophyte Neotyphodium lolii. This particular endophyte is asexual and has the potential to impact host survival, growth and reproduction. The objective was to assess the potential costs or benefits of endophyte infection on drought stress tolerance of native perennial ryegrass accessions originally collected from Italy, Morocco, Tunisia, and Turkey. Sixty infected (E+) individuals from each accession were planted in a greenhouse. Half of these individuals were treated with a systemic fungicide to eliminate the endophyte (E−). For two drought periods water was withheld for 10-14 days and then allowed a one week recovery period following each. In some accessions under drought, E+ plants had more tillers, greater tiller lengths, total dry mass and green shoot mass than E− plants, suggesting a positive effect of endophyte infection on host growth. Total tiller length and the number of tillers showed significant population × treatment × infection interactions for 4 of 6 populations. This work is one of the few that documents the effects of endophyte infection for a common forage grass species from wild populations native to its distributional range. The results demonstrate that endophyte infection can help ameliorate abiotic stress such as drought and there may be a selective advantage for grasses from certain Mediterranean regions.     

胡杨异形叶叶绿素荧光特性对高温的响应

胡杨(Populus euphratica Oliv)是塔里木极端干旱荒漠区优势乔木树种,由于其生长在荒漠环境中,极端高温远高于其它地区,因此研究胡杨对高温胁迫的响应特征对于解释胡杨的抗逆机理与生态适应策略具有极为重要的意义。以胡杨3种典型的异形叶为材料,研究不同温度对其叶绿素荧光特性和能量分配的影响。结果表明,25-45 ℃温度下胡杨异形叶的F₀、F_m、F_v/F_m与F_v/F₀变化不大,尤其F_v/F_m仍能保持在0.78左右,光合反应正常;高温胁迫下(＞45 ℃)F_m、F_v/F₀、F_v/F_m、F'_v/F'_m、qP、ΦPSⅡ、P和ETR均大幅降低;F₀、qN、E显著上升,而D先上升后下降,说明高温抑制了PSⅡ的功能,使PSⅡ反应中心活性下降,QA^-的还原速率加快,光化学电子传递速率降低,某些能量耗散途径受阻,影响了PSⅠ和PSⅡ激发能的平衡分配,最终导致光合机构受损、光合速率降低。胡杨3种异形叶的叶绿素荧光参数随温度升高变幅不同,高温处理下锯齿阔卵形叶各参数均高于卵形叶与条形叶,表明锯齿阔卵形叶比卵形叶和条形叶具有更强的高温耐受能力。用模糊数学的隶属度函数对胡杨3种异形叶的耐热性进行综合评价,锯齿阔卵形叶的耐热性最强。     

VfCPK1, a gene encoding calcium-dependent protein kinase from Vicia faba, is induced by drought and abscisic acid

Calcium, one of the most ubiquitous second messengers, has been shown to be involved in a wide variety of responses in plants. Calcium-dependent protein kinases (CDPKs) (EC 2.7.1.37) are the predominant Ca(2+)-regulated serine/threonine protein kinase in plants and play an important role in plant calcium signal transduction. CDPKs are encoded by a large multigene family in many plants, which has been showed so far; however, the precise role of each specific CDPK is still largely unknown. A novel CDPK gene designated as VfCPK1 was cloned from epidermal peels of broad bean (Vicia faba L.) leaves using the rapid amplification of cDNA ends (RACE)-PCR technique and its expression was studied in detail. The VfCPK1 cDNA is 1783 bp long and contains an open reading frame of 1482 bp encoding 493 amino acids. VfCPK1 contains all conserved regions found in CDPKs and shows a high level of sequence similarity to many other plant CDPKs. VfCPK1 was highly expressed in leaves, especially in leaf epidermal peels of broad bean in mRNA and protein levels. Expressions of VfCPK1 at both the mRNA and protein levels were increased in leaves treated with abscisic acid or subjected to drought stress. Potential roles of VfCPK1 in epidermal peels are discussed. The nucleotide sequence data reported here were deposited in the GenBank database under accession number AY753552.     

A salt-inducible chloroplastic monodehydroascorbate reductase from halophyte Avicennia marina confers salt stress tolerance on transgenic plants

Plant growth and productivity are adversely affected by various abiotic stress factors. In our previous study, we used Avicennia marina, a halophytic mangrove, as a model plant system for isolating genes functioning in salt stress tolerance. A large scale random EST sequencing from a salt stressed leaf tissue cDNA library of one month old A. marina plants resulted in identification of a clone showing maximum homology to Monodehydroascorbate reductase (Am-MDAR). MDAR plays a key role in regeneration of ascorbate from monodehydroascorbate for ROS scavenging. In this paper, we report the cellular localization and the ability to confer salt stress tolerance in transgenic tobacco of this salt inducible Am-MDAR. A transit peptide at the N-terminal region of Am-MDAR suggested that it encodes a chloroplastic isoform. The chloroplastic localization was confirmed by stable transformation and expression of the Am-MDAR-GFP fusion protein in tobacco. Transgenic tobacco plants overexpressing Am-MDAR survived better under conditions of salt stress compared to untransformed control plants. Assays of enzymes involved in ascorbate–glutathione cycle revealed an enhanced activity of MDAR and ascorbate peroxidase whereas the activity of dehyroascorbate reductase was reduced under salt stressed and unstressed conditions in Am-MDAR transgenic lines. The transgenic lines showed an enhanced redox state of ascorbate and reduced levels of malondialdehyde indicating its enhanced tolerance to oxidative stress. The results of our studies could be used as a starting point for genetic engineering of economically important plants tolerant to salt stress.     

Knock-out of Arabidopsis AtNHX4 gene enhances tolerance to salt stress

AtNHX4 belongs to the monovalent cation:proton antiporter-1 (CPA1) family in Arabidopsis. Several members of this family have been shown to be critical for plant responses to abiotic stress, but little is known on the biological functions of AtNHX4. Here, we provide the evidence that AtNHX4 plays important roles in Arabidopsis responses to salt stress. Expression of AtNHX4 was responsive to salt stress and abscisic acid. Experiments with CFP-AtNHX4 fusion protein indicated that AtNHX4 is vacuolar localized. The nhx4 mutant showed enhanced tolerance to salt stress, and lower Na⁺ content under high NaCl stress compared with wild-type plants. Furthermore, heterologous expression of AtNHX4 in Escherichia coli BL21 rendered the transformants hypersensitive to NaCl. Deletion of the hydrophilic C-terminus of AtNHX4 dramatically increased the hypersensitivity of transformants, indicating that AtNHX4 may function in Na⁺ homeostasis in plant cell, and its C-terminus plays a role in regulating the AtNHX4 activity.     

胡杨小孢子发生及微管骨架变化与异常研究

利用压片法和间接免疫荧光结合DAPI(4′,6-diamidino-2-phenylindole)染色法,对胡杨小孢子母细胞减数分裂过程中微管骨架变化和染色体行为进行观察研究。结果表明:（1）胡杨小胞子母细胞减数分裂进程中染色体行为正常,其中:偶线期可观察到单价体,中期Ⅰ会出现落后染色体,末期Ⅰ和末期Ⅱ的核仁呈现动态变化。（2）胡杨小孢子发生过程中细胞内微管骨架呈动态变化过程,其中:中期Ⅱ形成平行纺锤体以及三极纺锤体;末期Ⅱ未观察到典型的成膜体结构,同时型胞质分裂受子核间辐射微管系统调节,通过胞质向心收缩而发生,胞质分裂后形成四边形和四面体型四分体。（3）胡杨小孢子母细胞减数分裂过程中还存在各种异常细胞学现象,其中:中期Ⅱ平行纺锤体发生融合;中期Ⅱ 和后期Ⅱ孢母细胞两个纺锤体间的胞质会出现裂沟;四分体时期存在三分体和二分体,并产生天然2n花粉和连体花粉。