How modeling can reconcile apparently discrepant experimental results: The case of pacemaking in dopaminergic neurons

Midbrain dopaminergic neurons are endowed with endogenous slow pacemaking properties. In recent years, many different groups have studied the basis for this phenomenon, often with conflicting conclusions. In particular, the role of a slowly-inactivating L-type calcium channel in the depolarizing phase between spikes is controversial, and the analysis of slow oscillatory potential (SOP) recordings during the blockade of sodium channels has led to conflicting conclusions. Based on a minimal model of a dopaminergic neuron, our analysis suggests that the same experimental protocol may lead to drastically different observations in almost identical neurons. For example, complete L-type calcium channel blockade eliminates spontaneous firing or has almost no effect in two neurons differing by less than 1% in their maximal sodium conductance. The same prediction can be reproduced in a state of the art detailed model of a dopaminergic neuron. Some of these predictions are confirmed experimentally using single-cell recordings in brain slices. Our minimal model exhibits SOPs when sodium channels are blocked, these SOPs being uncorrelated with the spiking activity, as has been shown experimentally. We also show that block of a specific conductance (in this case, the SK conductance) can have a different effect on these two oscillatory behaviors (pacemaking and SOPs), despite the fact that they have the same initiating mechanism. These results highlight the fact that computational approaches, besides their well known confirmatory and predictive interests in neurophysiology, may also be useful to resolve apparent discrepancies between experimental results. © 2011 Drion et al.

Otolith characteristics and age determination of an endemic Ptychobarbus dipogon (Regan, 1905) (Cyprinidae: Schizothoracinae) in the Yarlung Tsangpo River, Tibet

We describe the microincrements, checks and annuli in the lapilli of the schizothoracine Ptychobarbus dipogon, an endemic species of the Tibetan plateau. We collected samples in the Yarlung Tsangpo River and its tributaries on a monthly basis (from April 2004 to August 2006). We describe the shape features of the three pairs of otoliths and document the full trajectory of lapillus development. We found that five to seven checks were clearly visible in the opaque zone of the first annulus. The pattern of 21-23 daily growth increments within each check might be explained as a lunar-induced deposition. We counted between 137 and 154 increments within the first annulus. Annuli appeared as a sequence of gradually declining increment widths, whereas false rings were characterized by abrupt checks. Our oldest estimates were 23(+)years for males and 44(+) for females. The time of annulus completion was clearly between March and April each year using monthly marginal increments analysis. We consider the factors responsible for daily increment formation as an endogenous circadian rhythm. Environmental information, such as strong sunlight and cold water temperatures in the Tibetan Plateau, could reinforce the endogenous daily cycle. Our results provided important data addressing the ecology and population dynamics of P. dipogon.

Molecular cloning and expression characterization of ApoC-I in the orange-spotted grouper

Endogenous yolk nutrients are crucial for embryo and larval development in fish, but developmental behavior of the genes that control yolk utilization remains unknown. Apolipoproteins have been shown to play important roles in lipid transport and uptake through the circulation system. In this study, EcApoC-I, the first cloned ApoC-I in teleosts, has been screened from pituitary cDNA library of female orange-spotted grouper (Epinephelus coioides), and the deduced amino acid sequence shows 43.5% identity to one zebrafish (Danio rerio) hypothetical protein similar to ApoC-I, and 21.2%, 21.7%, 22.5%, 20%, and 22.5% identities to Apo C-I of human (Homo sapiens), house mouse (Mus musculus), common tree shrew (Tupaia glis), dog (Canis lupus familiaris) and hamadryas baboon (Papio hamadryas), respectively. Although the sequence identity is low, amphipathic alpha-helices with the potential to bind to lipid were predicted to exist in the EcApoC-I. RT-PCR analysis revealed that it was first transcribed in gastrula embryos and maintained a relatively stable expression level during the following embryogenesis. During embryonic and early larval development, a very high level of EcApoC-I expression was in the yolk syncytial layer, indicating that it plays a significant role in yolk degradation and transfers nutrition to the embryo and early larva. By the day 7 after hatching, EcApoC-I transcripts were observed in brain. In adult, EcApoC-I mRNA was detected abundantly in brain and gonad. In transitional gonads, the EcApoC-I expression is restricted to the germ cells. The data suggested that EcApoC-I might play an important role in brain and gonad morphogenesis and growth.

Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

The response of antioxidant systems in Nostoc sphaeroides against UV-B radiation and the protective effects of exogenous antioxidants

UV radiation is one of many harmful factors found in space that are detrimental to organisms on earth in space exploration. In the present work, we examined the role of antioxidant system in Nostoc sphaeroides Kutz (Cyanobacterium) and the effects of exogenously applied antioxidant molecules on its photosynthetic rate under UV-B radiation. It was found that UV-B radiation promoted the activity of antioxidant system to protect photosystem 11 (PSII) and exogenously applied antioxidant: sodium nitroprusside (SNP) and N-acetylcysteine (NAC) had an obvious protection on PSII activity under UV-B radiation. The activity of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and content of NIDA (malondialdehyde) and ASC (ascorbate) were improved by 0.5 mM and 1 mM SNP, but 0.1 mM SNP decreased the activity of antioxidant system. Addition of exogenous NAC decreased the activity of SOD, POD, CAT and the content MDA and ASC. In contrast, exogenously applied NAC increased GSH content. The results suggest that exogenous SNP and NAC may protect algae by different mechanisms: SNP may play double roles as both sources of reactive free radicals as well as ROS scavengers in mediating the protective role of PSII on algae under UV-B radiation. On the other hand, NAC functions as an antioxidant or precursor of glutathione, which could protect PSII directly from UV-B radiation. (c) 2007 COSPAR, Published by Elsevier Ltd. All rights reserved.

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F-1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P-0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Efficient RNA interference in zebrafish embryos using siRNA synthesized with SP6 RNA polymerase

Double-stranded RNA (dsRNA) has been shown to be a useful tool for silencing genes in zebrafish (Danio rerio), while the blocking specificity of dsRNA is still of major concern for application. It was reported that siRNA (small interfering RNA) prepared by endoribonuclease digestion (esiRNA) could efficiently silence endogenous gene expression in mammalian embryos. To test whether esiRNA could work in zebrafish, we utilized Escherichia coli RNaseIII to digest dsRNA of zebrafish no tail (ntl), a mesoderm determinant in zebrafish and found that esi-ntl could lead to developmental defects, however, the effective dose was so close to the toxic dose that esi-ntl often led to non-specific developmental defects. Consequently, we utilized SP6 RNA polymerase to produce si-ntl, siRNA designed against ntl, by in vitro transcription. By injecting in vitro synthesized si-ntl into zebrafish zygotes, we obtained specific phenocopies of reported mutants of ntl. We achieved up to a 59%no tail phenotype when the injection concentration was as high as 4 mu g/mu L. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization analysis showed that si-ntl could largely and specifically reduce mRNA levels of the ntl gene. As a result, our data indicate that esiRNA is unable to cause specific developmental defects in zebrafish, while siRNA should be an alternative for downregulation of specific gene expression in zebrafish in cases where RNAi techniques are applied to zebrafish reverse genetics.

Evaluation of soot particles of biomass fuels with endocrine-modulating activity in yeast-based bioassay

Exposure to indoor air pollution (IAP) from the combustion of biomass fuels is an important cause of morbidity and mortality in developing countries. In the work discussed in this paper we evaluated the endocrine activity of soot particles from biomass fuels by using yeast bioassay. These pollutants could have beta-galactosidase activity with a relative potency (RP) about 10(-7)-10(-9) that of estradiol. Soot particles from wood and straw combustion only partially induced beta-galactosidase activity whereas others produced fully inductive activity in the yeast assay system. These pollutants did not have estrogen antagonist and progesterone agonist activity within the defined concentration range. However, these pollutants require 2-4 orders of magnitude higher IC50 to inhibit the activity of progesterone in a similar dose-response manner to mifepristone. We therefore propose that the endocrine activity of some environmental pollutants may be because of inhibition of the progesterone receptor (hPR). GC-MS results showed that substituted polycyclic aromatic hydrocarbon (PAH) compounds, substituted phenolic compounds and derivatives, aromatic carbonyl compounds, and phytosteroids in these soot particles may be mimicking endogenous hormones.

玫瑰花芽分化期矿质元素和内源激素的变化规律

本文以沈阳市于洪区玫瑰种植示范区内多季花冷香玫瑰和单季花平阴玫瑰为研究对象，通过野外采样并测定了两者在花芽分化期矿质元素（P、K、Ca、Mg、Fe、Zn、B、Mn、Cu）和内源激素（IAA、GA、ZR、ABA）的含量，并对其含量及比值变化进行分析，初步确定了玫瑰花芽分化与元素、激素之间的相互关系。研究结果将为玫瑰花期的化学调控、提高产花量和正确制定栽培技术措施提供理论依据，也为玫瑰进一步开发利用打下基础。主要结论：1．在冷香玫瑰和平阴玫瑰的整个花芽分化期，P、K、Cu、Zn大体上均呈下降趋势，表明它们可能参与玫瑰成花；ca、Mg在冷香玫瑰和平阴玫瑰的花蕾形成期含量都下降，可能意味着ca、Mg参与两者的花蕾形成；Fe与玫瑰花芽分化关系不大；高含量的B和Mn有利于两者花蕾形成。2．在冷香玫瑰和平阴玫瑰花蕾形成期，IAA和GA出现低水平，ZR和ABA出现高水平，激素比值（ABA/GA，ABA／IAA，ZR/GA，ZR/IAA）出现高水平，表明ABA和ZR促进两者开花；而IAA和GA抑制两者开花；高比例的（ABA/GA，ABA/IAA，ZR/GA，ZR/IAA）有利于两者开花。

遮荫与外源脱落酸喷施对青杨(Populus cathayana Rehd.)不同种群抗旱性的影响

杨树具有分布广、适应性强的特征，在生态环境治理和解决木材短缺方面均占有重要位置。青杨(Populus cathayana Rehd.)是青杨派树种的重要成员之一，也是生长较迅速、易繁殖的重要杨树资源。本研究选取了来自不同气候地区的青杨两种群为材料，采用植物生态学、生理学和生物化学的研究方法，系统地研究了青杨对干旱与遮荫、干旱与外源脱落酸(ABA)喷施的生长、形态、生理和生化响应及种群间差异，研究成果可为我国干旱半干旱地区的造林以及生态恢复提供理论依据和科学指导。主要研究结论如下：1.青杨在干旱胁迫下的适应机制为：生长性状及生物量的分配变化：干旱胁迫下虽然植株生长受抑，株高、基茎及各部分生物量都显著减小，但有相对较多的生物量向根部分配，根/冠比以及细/粗根比增加。青杨对干旱胁迫的光合作用表现为：干旱胁迫降低了青杨的净光合速率、蒸腾速率、气孔导度以及光合氮利用效率，提高了瞬时用水效率。干旱还引起了活性氧的产生，使得膜脂过氧化产物丙二醛(MDA)增加，同时也增强了植物抗氧化酶系统(如超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性的增加)及非酶系统的能力(如抗坏血酸(AsA)含量的增加)。干旱降低了植物叶片的相对含水量，而促进了渗透调节物质(游离脯氨酸及可溶性糖)的积累，增加了植物的渗调能力。干旱下青杨两种群的内源ABA含量显著增加，碳同位素组分(δ13C)也显著提高。这些结果证明植物遭受干旱胁迫后发生一系列的形态、生理和生化响应，这些变化能提高植物在干旱下的存活和生长能力。2.青杨两种群对干旱胁迫反应的种群差异：与来自湿润地区的汉源种群相比，来自干旱地区的乐都种群在干旱条件下生物量向根系分配的可塑性更强，同时具有更强的抗氧化系统能力，所受到活性氧的伤害也更少，并且累积更多的脯胺酸和ABA，具有更高的δ13C。这些都说明了乐都种群对干旱的适应性比汉源种群更强。两种群对干旱的响应差异应归于它们的用水策略的不同：汉源种群来自湿润地区，采用了耗水型的用水策略，抗旱能力较弱；而乐都种群，来自干旱地区，通常采用节水型的用水策略，有更强的抗旱能力。3.遮荫对青杨两种群抗旱性的影响：遮荫对青杨抗旱性的影响决定于遮荫程度的不同，我们的结果表明中度的遮荫可以有效的提高干旱下植物的生长，对干旱胁迫有明显的缓解作用，具体体现在中度遮荫下受旱植物的叶片相对含水量得到提高，使得植物体内水分状况得到了改善；光合速率并未降低，植物光合氮利用效率增加，说明中度的遮荫并未明显限制植物的碳获得；抗氧化酶活性与膜脂过氧化产物MDA含量的同时降低，说明中度遮荫下所受到的活性氧伤害减少；中度遮荫下的ABA及δ13C的变化也不如在全光下变化明显，这也说明中度遮荫缓解了干旱胁迫。但是重度的遮荫却对干旱胁迫有明显的加剧作用，主要表现在重度遮荫降低了植物的光合速率，严重抑制了植物的生长；同时重度遮荫下脯胺酸含量和抗氧化酶活性的急剧下降，导致了植物渗调能力的下降及膜脂过氧化产物MDA的显著升高；重度遮荫还显著降低了内源ABA的累积和δ13C，降低了植物的抗旱能力。此外，青杨两种群在对干旱和遮荫的响应中，也表现出种群差异。汉源种群，来自湿润且年日照辐射较少的地区，表现出相对更强的耐荫性和需水性。而乐都种群，来自干旱且年日照辐射丰富的地区，表现出相对更强的耐旱性和需光性。这说明了植物对环境胁迫的耐受性是其长期适应原生境的结果，并且来自不同气候地区的两种群在面临环境胁迫时会采取不同的生存策略。4. 外源ABA喷施对青杨两种群抗旱性的影响：外源ABA的喷施可以提高两种群的抗旱性，具体表现为外源ABA喷施促进了青杨根系的生长，显著提高了干旱下植物的根/冠比和细/粗根比，减少了比叶面积；在生理生化方面，外源ABA降低了干旱下植物叶片的气孔导度，降低了蒸腾速率和净光合速率，但提高了瞬时用水效率，提高了叶片的相对含水量，增加了干旱下植物的保水能力。外源ABA进一步增加了干旱下植物内源ABA的积累，促进了植物渗调物质如脯胺酸和可溶性糖的积累，增加了抗氧化酶系统(如SOD、APX、CAT)的活性和非酶系统AsA的含量，降低了活性氧(如超氧阴离子(O2和过氧化氢(H2O2))对植株的伤害。此外，外源ABA还进一步提高了干旱下植物的δ13C，提高了植物的长期用水效率，由此提高了植物的抗旱能力。另一方面，两种群对外源ABA和干旱的响应也有所差别。来自湿润地区的汉源种群，对干旱较为敏感，所受干旱的影响也较大，而外源ABA的喷施对汉源种群抗旱性的提高作用也更为突出。乐都种群，由于其长期适应干旱地区的生长，本身已具有较强的抗旱能力，因此外源ABA喷施对其抗旱性的提高不如对汉源种群的效果明显。由此我们可以得出对于一些抗性弱或干旱敏感的物种或者种群，可以采用外施ABA的方法来提高其抗性。Poplars play an important role in lumber supply, and are important component ofecosystems due to their wide distribution and well adaptation. Populus cathayana Rehd.,which belongs to Populus Sect. Tacamahaca Spach, is one of the most important resources ofpoplars for its fast growth and reproductive. In this study, different populations of P.cathayana were used as experiment material to investigate the adaptability to drought stressand population differences in adaptability, and the effects of shade and exogenous abscisicacid (ABA) application on the drought tolerance. Our results could provide a strongtheoretical evidence and scientific direction for the afforestation, and rehabilitation ofecosystem in the arid and semi-arid area, and provide a strong evidence for adaptivedifferentiation of different populations, and so may be used as criteria for species selectionand tree improvement. The results are as follows:1. A large set of parallel response to drought stress: Drought stress caused pronouncedinhibition of the growth and increased relatively dry matter allocation into the root. For thetwo populations, the shoot height, basal diameter and total biomass were decreased but theroot/shoot ratio and fine root/coarse root ratio were increased under drought conditions；Drought stress caused pronounced inhibition of photosynthesis, decreased the stomatalconductance, transpiration rate, and photosynthetic nitrogen-use efficiency (PNUE) butincreased the instantaneous water use efficiency. Drought significantly improved the levels ofreactive oxygen species and malondialdehyde (MDA) and to induce the entire set ofantioxidative systems including the increase of activities of superoxide dismutase (SOD),ascorbate peroxidase (APX), catalase (CAT) and ascorbate (AsA) content. Drought decreased the leaf relative water content (RWC) but improved the capability of osmotic adjustmentindicated by the higher proline accumulation. Drought also increased the ABA content andcarbon isotope composition (δ13C), which indicating the long period water use efficiency wasimproved under drought. These results demonstrate that there are a large set of parallelchanges in the morphological, physiological and biochemical responses when plants areexposed to drought stress; these changes may enhance the capability of plants to survive andgrow during drought periods.2. Difference in adaptation to drought stress between contrasting populations of P.cathayana: Compared with the Hanyuan population (wet climate), the Ledu population (dryclimate) showed higher root/shoot ratio and water use efficiency, exhibited higherantioxidative systems capability thus resulting in less oxidative damage, accumulated moreABA and free proline content under drought conditions. The results suggested that there weredifferent water-use strategies between the two populations. The Ledu population, whichcomes from dry climate region, with higher drought tolerance, may employ a conservativewater-use strategy, whereas the Hanyuan population, which comes from wet climate, withlower drought tolerance, may employ a prodigal water-use strategy. These variations indrought responses may be used as criteria for species selection and tree improvement.3. The effects of shade on the drought tolerance: The reduction in the availability of lightand water affected the morphological and physiological responses of the two P. cathayanapopulations. In addition, the light environment modified the growth responses of P.cathayana seedlings to varying water environments in different ways depending upon theintensity of the light levels considered. There is an apparent alleviation to drought effects bymoderate shade in P. cathayana seedlings, as indicated by the higher leaf RWC, and unchanged net photosynthesis and PNUE, as well as by the lower antioxditative enzymeactivity, MDA, ABA and δ13C levels, which implied moderate shade did not significantlylimited the carbon acquisition or inhibited the plant growth, but ameliorated the detrimentaleffects of drought. On the other hand, an apparent aggravation to drought effects by severeshade was also observed, as indicated by the pronounced decrease of plant growth and net photosynthesis, the lower total biomass, ABA level, δ13C, free proline content andantioxditative enzyme activity and higher MDA accumulation. By contrast, the twopopulations showed different responses to shade and drought. The Hanyuan population,which comes from a riparian basin having a relatively wet climate and less annual solarradiation, is more sensitive to drought but more tolerant to shade. The Ledu population, whichcomes from a mountainous plateau with less rainfall and with more annual solar radiation, ismore tolerant to drought but more sensitive to shade. The results demonstrated that theendurance of plants to stress is a result of long-term evolution and adaptation to theenvironment, as suggested by the different strategies employed by the P. cathayanapopulations originating from contrasting habitats when they were exposed to drought andshade.4. The effects of exogenous ABA application on the drought tolerance: For bothpopulations under drought conditions tested, exogenous ABA application significantlyimproved the root/shoot ratio, fine root/coarse root ratio, and decreased the specifical leaf area.On the physiological and biochemical traits, exogenous ABA application significantlydecreased stomatal conductance, transpiration rate and net photosythesis but increased theinstance water use efficiency and leaf RWC. On the other hand, exogenous ABA applicationsignificantly increased endogenous ABA, proline, solube sugar and AsA content, as well asSOD, APX and CAT activities, thus reduced the damage of reactive oxygen species. Moreover,the long period water use efficiency as indicated by δ13C was also improved by exogenousABA application. In additionally, there was different responsive between the two populationsto drought and exogenous ABA application. The Hanyuan population, which comes from wetclimate region, is more sensitive to drought, and the effect of exogenous ABA is moreobviously than that in the Ledu population, which comes from dry climate region and is moredrought-responsive. Therefore, we can use exogenous ABA application to improve theresistance of plants, especially for the drought- sensitive species or populations.

增强UV-B辐射对粗枝云杉(Picea asperata Mast.)和青杨(Populus cathayana Rehd.)的影响

光是植物赖以生存的重要环境因子，但是植物在获得光的同时不可避免的会受到紫外辐射的伤害。尤其是近年来，人类向大气中排放的大量氮氧化合物和氟氯烃类化合物(CFC’s)引起臭氧分子的分解，导致到达地球表面的紫外辐射增加，特别是UV-B辐射增强。而另一方面，植物对UV-B辐射反应的敏感性在种间和品种间存在差异，主要受植物基因型，生态型和生活型的控制。本项目分别以粗枝云杉和青杨组杨树为模式植物，从形态和生理生化方面分别研究了来自不同水分背景下的粗枝云杉种群和来自不同UV-B背景下的青杨种群在增强UV-B下的反应及其反应差异,并探讨了干旱、喷施外源脱落酸(ABA)对它们抗UV-B能力的影响。研究成果可为生态系统的恢复与重建提供理论依据和科学指导。主要研究结果如下： 1. 粗枝云杉的两个种群，湿润种群(来自四川黑水)和干旱种群(来自甘肃迭部)在水分良好和干旱状况下表现出对增强UV-B的不同响应。同时，干旱对粗枝云杉抗UV-B能力的影响也得到研究：两种胁迫共同作用时，干旱表现出在一定程度上减弱了增强UV-B对粗枝云杉的生理特性的影响。 干旱胁迫显著降低了两个粗枝云杉种群的光合同化速率(A), 气孔导度(gs)和PSII的有效光量子产量(Y), 同时，提高了非光化学猝灭效率(qN)和超氧化物歧化酶(SOD)的活性。与湿润种群相比，干旱种群抗旱性更强，表现为干旱种群拥有更高的SOD和干旱进一步加剧了UV-B的胁迫效应。 本研究中，干旱胁迫单独作用时，显著降低了青杨两个种群的生物量积累和气体交换，具体包括A、gs、蒸腾速率(E)和光合氮利用效率(PNUE)，提高了两个种群的瞬时水分利用效率(WUEi)、长期水分利用效率(WUET)、碳同位素组分(δ13C)和氮含量(N)。同时，UV吸收物质和ABA含量也得到积累。另一方面，增强UV-B对青杨两个种群各个指标的影响，同干旱所引起的效应有着相似的趋势。同低海拔种群相比，高海拔种群有着更强的抗旱和抗UV-B能力，具体表现在高海拔种群有着更多的生物量积累，更强的气体交换和水分利用效率及更高水平的ABA和UV吸收物质含量。相比干旱诱导的生物量积累和气体交换的降低，在干旱和增强UV-B两个胁迫同时作用于青杨时，这种降低表现的更为明显。显著的干旱和UV-B的交互作用还表现在WUEi, WUET, δ13C, 可溶性蛋白含量, UV吸收物质含量, ABA, 叶片和茎中的N含量以及C/N比中。 3. 经过一个生长季的试验观察，增强UV-B、外源ABA及两因子共同作用对青杨的生物量积累、气体交换、内源ABA和UV吸收物质含量、抗氧化系统以及碳、氮含量和碳/氮比均产生显著影响。本试验中，青杨的两个种群分别来自中国西南部的不同海拔地区，高海拔种群来自青海大通而低海拔种群来自四川九寨。外源ABA的胁迫为直接喷施ABA到青杨叶片，而增强UV-B胁迫是利用平方波系统分别保证青杨苗暴露于外界UV-B强度和两倍于外界UV-B强度下。 研究结果显示，增强UV-B显著的降低了两个青杨种群的株高、基茎、总叶面积和总生物量等生长指标，同时也导致其A、gs、E和叶片中碳含量的减少。而显著增加了SOD和过氧化物酶(GPx)活性水平，诱导了过氧化氢(H2O2)和MDA的显著增加，促进了UV吸收物质和不同器官中内源ABA含量的显著积累。另一方面，外源ABA引起了青杨光合同化速率的下降，SOD和GPx酶活性的增强，H2O2 和 MDA含量也表现出显著增加，同时，内源ABA含量得到显著累积。同低海拔种群相比，高海拔种群具有更加抗UV-B和外源ABA的特性。显著的UV-B和ABA的交互作用表现在A, E, SOD和GPx活性，以及叶片和根部的内源ABA等一系列指标中。在所有胁迫下，叶片中的碳和氮含量同其在茎和根中的含量显著相关，另外，叶片和茎中的氮含量同茎中的碳含量显著相关。 Sunlight is an indispensable environment factor for plants survival and development. Meanwhile, photosynthetic organisms need sunlight and are thus, inevitably, exposed to UV radiation. Especially for recent years, ultraviolet radiation, especially UV-B reaching the Earth’s surface increased because of depletion of ozone layer resulted from emission of NxO and CFC’s from human activities. On the other hand, the sensitivity of plants to UV-B radiation depends on the species, developmental stage and experimental conditions. In this experiment, two populations of Picea asperata Mast from different water background and two populations of Populus cathayana Rehder from different altitude background were selected as model plants to assess the effects of enhanced UV-B radiation. Morphological and physiological traits induced by enhanced UV-B in each plant species were observed and the different responses were discussed, furthermore the influences of drought and exogenous ABA on responses induced by enhanced UV-B were studied. The study could provide a strong theoretical evidence and scientific direction for the afforestation and rehabilitation of ecosystem. The results are as follows: 1. Different responses of two contrasting Picea asperata Mast. populations to enhanced ultraviolet-B (UV-B) radiation under well-watered and drought conditions were investigated. And the effects of enhanced UV-B on tolerance of drought were also observed in our study that the UV-B exposure may have alleviated some of the damage induced by drought. Two contrasting populations, originating from a wet and dry climate region in China, respectively, were employed in our study. Drought significantly decreased CO2 assimilation rate (A), stomatal conductance (gs) and effective PSII quantum yield (Y), while it significantly increased non-photochemical quenching (qN) and the activity of superoxide dismutase (SOD) in both populations. Compared with the wet climate population, the dry climate population was more acclimated to drought stress and showed much higher activities of SOD and ascorbate peroxidase (APX), and much lower levels of malondialdehyde (MDA) and electrolyte leakage. On the other hand, enhanced UV-B radiation also induced a significant decrease in the chlorophyll (Chl) content in both populations under well-watered conditions, and a significant increase in UV-absorbing compounds in the wet climate population. After one growing season of exposure to different UV-B levels and watering regimes, the increases in MDA and electrolyte leakage, as induced by drought, were less pronounced under the combination of UV-B and drought. In addition, an additive effect of drought and UV-B on A and gs was observed in the wet climate population, and on the activity of APX and qN in the dry climate population. 2. The significant effects of drought, enhanced UV-B radiation and their combination on Populus cathayana Rehd. growth and physiological traits were investigated in two populations, originating from high and low altitudes in south-west China. Our results showed that UV-B acts as an important signal allowing P. cathayana seedlings to respond to drought and that the combination of drought and UV-B may cause synergistically detrimental effects on plant growth in both populations. In both populations, drought significantly decreased biomass accumulation and gas exchange parameters, including A, gs, E and photosynthetic nitrogen use efficiency (PNUE). However, instantaneous water use efficiency (WUEi), transpiration efficiency (WUET), carbon isotope composition (δ13C) and nitrogen (N) content, as well as the accumulation of soluble protein, UV-absorbing compounds and abscisic acid (ABA) were significantly increased by drought. On the other hand, cuttings from both populations, when kept under enhanced UV-B radiation conditions, showed very similar changes in all above-mentioned parameters, as induced by drought. Compared with the low altitude population, the high altitude population was more tolerant to drought and enhanced UV-B, as indicated by the higher level of biomass accumulation, gas exchange, water-use efficiency, ABA concentration and UV-absorbing compounds. After one growing season of exposure to different UV-B levels and watering regimes, the decrease in biomass accumulation and gas exchange, induced by drought, was more pronounced under the combination of UV-B and drought. Significant interactions between drought and UV-B were observed in WUEi, WUET, δ13C, soluble protein, UV-absorbing compounds, ABA and in the leaf and stem N, as well as in the leaf and stem C/N ratio. 3. During one growing season, significant effects induced by enhanced UV-B radiation, exogenous ABA and their combination on biomass accumulation, gas exchange, endogenous ABA and UV-absorbing compounds concentrations, antioxidant system as well as carbon (C) content, nitrogen (N) content and C/N ratio were investigated in two contrasting Populus cathayana populations, originating from high and low altitudes in south-west China. Exogenous ABA was sprayed to the leaves and enhanced UV-B treatment was using a square-wave system to make the seedlings under ambient (1×) or twice ambient (2×) doses of biologically effective UV-B radiation (UV-BBE). Enhanced UV-B radiation significantly decreased height, basal diameter, total leaf area, total biomass, A, gs, E and carbon (C) content in leaves, and significantly increased activities of SOD and guaiacol peroxidase (GPx), hydrogen peroxide (H2O2) and malonaldehyde (MDA) content as well as the accumulation of UV-absorbing compounds and endogenous ABA concentrations among different organs in both populations. In contrast, exogenous ABA showed significant decrease in A and significant increases in activities of SOD and GPx, H2O2, MDA content and the endogenous ABA concentrations. Compared with the low altitude population, the high altitude population was more tolerant to enhanced UV-B and exogenous ABA. Significant interactions between UV-B and ABA were observed in A, E, activities of SOD and GPx, as well as in endogenous ABA in leaves and roots of both populations. Across all treatments, C and N content in leaves was strongly correlated with those were in stems and roots, respectively. Additionally, leaf and stem N content were significant correlated with stem C content.

簇毛麦（ Dasypyrum villosum）矮秆突变体的研究

株高是农作物的重要农艺性状之一，适度矮化有利于农作物的耐肥、抗倒、高产等。20世纪50年代，以日本的赤小麦为矮源的半矮秆小麦的培育和推广，使得世界粮食产量显著增长，被誉为“绿色革命”。迄今为止，已报到的麦类矮秆、半矮秆基因已达70多个，但由于某些矮源极度矮化或者矮化的同时伴随不利的农艺性状，使得真正运用于育种实践的矮源较少。因此，发掘和鉴定新的控制麦类作物株高的基因，开展株高基因定位、克隆及作用机理等方面的研究，对实现麦类作物株高的定向改良，具有重要的理论意义和应用价值。簇毛麦(Dasypyrum villosum，2n＝14，VV)是禾本科簇毛麦属一年生二倍体异花授粉植物，为栽培小麦的近缘属。本课题组在不同来源的簇毛麦杂交后代中发现了一株自然突变产生的矮秆突变体。观察分析了该突变体的生物学特性，对矮秆性状进行了遗传分析，对茎节细胞长度、花粉的活力进行了细胞学观察，考察了该突变体内源赤霉素含量及不同浓度外施赤霉素对突变体的作用，分析了赤霉素生物合成途径中的内根贝壳杉烯氧化酶（KO）和赤霉素20氧化酶(GA20ox)的转录水平，对赤霉素20氧化酶和赤霉素3-β羟化酶(GA3ox)进行了克隆和序列分析，并对GA20ox进行了原核表达和表达的组织特异性研究。主要研究结果如下：1. 该突变体与对照植株在苗期无差异，在拔节后期才表现出植株矮小，相对对照植株，节间伸长明显受到抑制，叶鞘长度基本不变。在成熟期，对照植株的平均株高为110cm，而突变株的平均株高为32cm，仅为对照植株的1/3 左右。除了株高变矮以外，在成熟后期，突变株还表现一定程度的早衰和雄性不育。I2-KI染色法观察花粉活力结果表明，对照植株花粉90%以上都是有活力的，而突变植株的花粉仅20%左右有活力。2. 突变株与对照植株的杂交F1代均表现正常株高，表明该突变性状为隐性突变。F1代植株相互授粉得到的168株F2代植株中，株高出现分离，正常株高（株高高于80cm）与矮秆植株（株高矮于40cm）的株数比为130：38，经卡方检验，其分离比符合3：1的分离比，因此推测该突变体属于单基因的隐性突变。3. 用ELISA方法检测突变株和对照植株的幼嫩种子中内源性生物活性赤霉素（GA1＋3）含量，结果表明突变株的赤霉素含量为36 ng/ml，而对照植株的赤霉素含量为900 ng/ml。对突变株外施赤霉素，发现矮秆突变株的株高和花粉育性均可得到恢复。这些结果表明该突变株为赤霉素缺陷型突变。4. 用荧光定量PCR方法比较突变株与对照植株中内根贝壳杉烯氧化酶和赤霉素20氧化酶的转录水平，结果表明突变株的KO转录水平比对照植株分别提高了6倍（苗期）和16倍（成熟期），突变株的GA20ox转录水平与对照植株在苗期无明显差异，在成熟期突变株较对照植株则提高了10倍左右。这些结果表明该矮秆突变体与赤霉素的生物合成途径密切相关，而且极有可能在赤霉素的生物合成途径早期就发生了改变。5. 以簇毛麦总基因组为模板，同源克隆了GenBank登录号为EU142950，RT-PCR分离克隆了簇毛麦的GA3ox基因cDNA全长序列，分析结果表明该cDNA全长1206bp，含完整编码区1104bp，推测该序列编码蛋白含368个氨基酸残基，分子量为40.063KD，等电点为6.27。预测的氨基酸序列含有双加氧酶的活性结构，在酶活性中心2个Fe离子结合的氨基酸残基非常保守。该序列与小麦、大麦和水稻的GA3ox基因一致性分别为98%、96%、86%。基因组序列与cDNA序列在外显子部分一致，在478-715bp和879-1019bp处分别含238bp和140bp的内含子。6. 通过RT-PCR技术克隆了簇毛麦的GA20ox基因全长，命名为DvGA20ox，GenBank登录号为EU142949。该基因全长1080个碱基，编码359个氨基酸，具有典型的植物GA20ox基因结构。该基因编码的蛋白质与小麦、大麦、黑麦草等GA20ox蛋白的同源性分别为98%，97% 和91%。该序列重组到原核表达载体pET-32a(+)上，将获得的重组子pET-32a(+)-DvGA20ox转化大肠杆菌BL21pLysS后用IPTG进行诱导表达。SDS-PAGE分析表明，DvGA20ox基因在大肠杆菌中获得了高效表达，融合蛋白分子量为55kDa。定量PCR分析表明，该基因在簇毛麦不同器官中的表达差异明显：叶片中表达水平最高，根部表达水平次之，茎部和穗中表达较弱。在外施赤霉素后，该基因的表达水平在两小时以后急剧下降，表明该基因的表达受自身的反馈调节。本研究结果认为，(1)该簇毛麦矮秆突变体为单基因的隐性突变；(2)该矮秆突变体为赤霉素敏感突变，内源赤霉素含量检测表明突变体的内源性赤霉素含量仅为对照植株的1/30；(3)荧光定量PCR结果表明突变株的赤霉素生物合成途径的关键酶基因表达水平比对照植株高，而且突变植株的赤霉素生物合成改变很可能发生在赤霉素生物合成途径的早期；(4)GA20ox有表达的组织特异性，且受到自身产物的反馈调节。 Plant height is an impotrant agronomic trait of triticeae crops.Semi-dwarf cropcultivars, including those of wheat, maize and rice, have significantly increased grainproduction that has been known as “green revolution”. The new dwarf varieties couldraise the harvest Index at the expense of straw biomass, and, at the sametime, improvelodging resistance and responsiveness to nitrogen fertilizer. Moreover, dwarf traits ofplant are crucial for elucidating mechanisms for plant growth and development aswell. In many plant species, various dwarf mutants have been isolated and theirmodles of inheritance and physiology also have been widely investigated.The causesfor their dwarf phenotypes were found to be associated with plant hormones,especially, gibberellins GAs.Dasypyrum villosum Candargy (syn.Haynaldia villosa) is a cross-pollinating,diploid (2n = 2x = 14) annual species that belongs to the tribe Triticeae. It is native toSouthern Europe and West Asia, especially the Caucasuses, and grows underconditions unfavorable to most cultivated crops. The genome of D. villosum,designated V by Sears, is considered an important donor of genes to wheat for improving powdery mildew resistance, take-all, eyespot, and plant and seed storageprotein content. A spontaneous dwarf mutant was found in D. villosum populations.The biological character and modles of inheritance of this dwarf mutant are studied.The cell length of stem cell is observed. The influence of extraneous gibberellin tothe dwarf mutant is also examined; the transcript level of key enzyme of gibberellinbiosynthesis pathway in mutant and control plants is compared. GA3ox and GA20oxare cloned and its expression pattern is researched.1. The dwarf mutant showed no difference with control plants at seedlingstage.At mature stage, the average height of control plants were 110cm and the dwarfplants were 33cm. The height of the mutant plant was only one third of the normalplants due to the shortened internodes. Cytology observation showed that theelongation of stem epidermal and the parenchyma cells were reduced. The dwarfmutant also shows partly male sterile. Pollen viability test indicates that more than80% of the pollen of the mutant is not viable.2. The inheritance modle of this dwarf mutant is studied. All The F1 plantsshowed normal phenotype indicating that the dwarfism is controlled by recessivealleles. Among the 168 F2 plants, there are 130 normal plants and 30 dwarf plants, thesegregation proportion accord with Mendel’s 3:1 segregation. We therefore proposethat this dwarf phenotype is controlled by a single recessive gene.3. Quantitative analyses of endogenous GA1+3 in the young seeds indicated thatthe content of GA1+3 was 36ng/ml in mutant plants and 900ng/ml in normal plants.The endogenous bioactive GA1+3 in mutant plants are only about 1/30 of that innormal plants. In addition, exogenously supplied GA3 could considerably restore themutant plant to normal phenotype. These results showed that this mutant wasdefective in the GA biosynthesis.4. More than ten enzymes are involved in GA biosynthesis. KO catalyzes thefirst cytochrome P450-mediated step in the gibberellin biosynthetic pathway and themutant of KO lead to a gibberellin-responsive dwarf mutant. GA20ox catalyze therate-limited steps so that their transcript level will influence the endogenous GAbiosynthesis and modifies plant architecture. The relative expression levels of genesencoding KO and GA20ox were quantified by real time PCR to assess whether thechanges in GA content correlated with the expression of GA metabolism genes andwhere the mutant occurred during the GA biosynthesis pathway. In mutant plants,the transcript levels of KO increased about 6-fold and 16-fold at the seedling stage and elongating stage respectively comparing with the normal plants. For theseedlings, there was no notable difference in the expression of GA20ox betweenmutant and normal plants. At the elongating stage, GA20ox transcript increased 10times in mutant plants, suggesting that the GA biosynthesis pathway in mutant plantshad changed from the early steps rather than the late steps.5. A full length cDNA of D. villosum gibberellin 3β-hydroxylase homology(designated as DvGA3ox) was isolated and consisted of 1206bp containing an openreading frame of 1104bp encoding 368 predicted amino acid residues. Identityanalysis showed that the gibberellin 3β-hydroxylase nucleotide sequence shared 98%,96% and 86% homology with that of wheat, barley and rice. The predicted peptidecontained the active-site Fe of known gibberellin 3β-hydroxylase and the regionhomologous to wheat, barley and Arabidopsis. The genomic clone of gibberellin3β-hydroxylase has two introns.6. The full-length cDNA of D. villosum gibberellin 20 oxidase (designated asDvGA20ox) was isolated and consisted of 1080-bp and encoded 359 amino acidresidues with a calculated mol wt of 42.46 KD. Comparative and bio-informaticsanalyses revealed that DvGA20ox had close similarity with GA20ox from otherspecies and contained a conserved LPWKET and NYYPXCQKP regions. Tissueexpression pattern analysis revealed DvGA20ox expressed in all the tissues that wereexamined and the highest expression of DvGA20ox in expanding leaves followed byroots. Heterologous expression of this cDNA clone in Escherichia coli gave a fusionprotein that about 55KD. Transcript levels of DvGA20ox dramatically reduced twohours after application of biologically active GA3, suggesting that the biosynthesis ofthis enzymes might be under feedback control.

Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures

Plant cell cultures have been suggested as a feasible technology for the production of a myriad of plant-derived metabolites. However, commercial application of plant cell culture has met limited success with only a handful of metabolites produced at the pilot- and commercial-scales. To improve the production of secondary metabolites in plant cell cultures, efforts have been devoted predominantly to the optimization of biosynthetic pathways by both process and genetic engineering approaches. Given that secondary metabolism includes-the synthesis. metabolism and catabolism of endogenous compounds by the specialized proteins, this review intends to draw attention to the manipulation and optimization of post-biosynthetic events that follow the formation of core metabolite structures in biosynthetic pathways. These post-biosynthetic events-the chemical and enzymatic modifications, transport, storage/secretion and catabolism/degradation have been largely unexplored in the past. Potential areas are identified where further research is needed to answer fundamental questions that have implications for advanced bioprocess design. Anthocyanin production by plant cell cultures is used as a case study for this discussion, as it presents a good example of compounds for which there are extensive research publications but still no commercial bioprocess. It is perceived that research on post-biosynthetic processes may lead to future opportunities for significant advances in commercial plant cell cultures. (C) 2002 Elsevier Science Inc. All rights reserved.