Functional Characterisation of the Oil Palm Type 3 Metallothionein-like Gene (<Emphasis Type="Italic">MT3-B</Emphasis>) Promoter

In silico analysis showed that the differentially expressed type 3 oil palm metallothionein-like genes MT3-A and MT3-B share at least 11 common putative promoter regulatory elements. The identified motifs include W-boxes, TATCCA element, binding element for cytokinin response regulators and pollen-specific elements. A high degree of conservation was observed in their genomic organisation where the coding regions are divided at two identical positions in both genes by two AT-rich introns. Promoter activity of the MT3-B gene was analysed using a transient assay by bombarding oil palm tissue slices with a β-glucuronidase (GUS) gene construct and a stable reporter assay by analysing GUS expression in transformed Arabidopsis thaliana plants. Transient expression analysis revealed MT3-B promoter activity in oil palm root tissues but not in fruit mesocarp at 12 weeks after anthesis and spear leaves. The T3 homozygous transgenic Arabidopsis plants, harbouring the MT3-B promoter/GUS construct, showed reporter activity in cotyledons and mature leaves with lower expression levels in root tissues. The expression levels in the roots of the T3 homozygous transgenic plants increased five- and 2.5-folds when treated with 80 μM of Zn²⁺ and Fe²⁺, respectively. Altogether, these results indicate that the MT3-A and MT3-B promoter activities may be regulated by a variety of abiotic factors and MT3-B promoter may potentially be manipulated for use in plant genetic engineering for induced synthesis of gene product.     

Involvement of nitric oxide and auxin in signal transduction of copper-induced morphological responses in Arabidopsis seedlings

Background and Aims

Plants are able to adapt to the environment dynamically through regulation of their growth and development. Excess copper (Cu²⁺), a toxic heavy metal, induces morphological alterations in plant organs; however, the underlying mechanisms are still unclear. With this in mind, the multiple signalling functions of nitric oxide (NO) in plant cells and its possible regulatory role and relationship with auxin were examined during Cu²⁺-induced morphological responses.

Methods

Endogenous auxin distribution was determined by microscopic observation of X-Gluc-stained DR5::GUS arabidopsis, and the levels of NO, superoxide and peroxynitrite were detected by fluorescence microscopy. As well as wild-type, NO-overproducer (nox1) and -deficient (nia1nia2 and nia1nia2noa1-2) arabidopsis plants were used.

Key Results

Cu²⁺ at a concentration of 50 µm resulted in a large reduction in cotyledon area and hypocotyl and primary root lengths, accompanied by an increase in auxin levels. In cotyledons, a low Cu²⁺ concentration promoted NO accumulation, which was arrested by nitric oxide synthase or nitrate reductase inhibitors. The 5-μm Cu²⁺-induced NO synthesis was not detectable in nia1nia2 or nia1nia2noa1-2 plants. In roots, Cu²⁺ caused a decrease of the NO level which was not associated with superoxide and peroxynitrite formation. Inhibition of auxin transport resulted in an increase in NO levels, while exogenous application of an NO donor reduced DR5::GUS expression. The elongation processes of nox1 were not sensitive to Cu²⁺, but NO-deficient plants showed diverse growth responses.

Conclusions

In plant organs, Cu²⁺ excess results in severe morphological responses during which the endogenous hormonal balance and signal transduction are affected. Auxin and NO negatively regulate each other''s level and NO intensifies the metal-induced cotyledon expansion, but mitigates elongation processes under Cu²⁺ exposure.     

Engineering greater aluminium resistance in wheat by over-expressing TaALMT1

MethodsParticle bombardment was used to transform wheat with TaALMT1, the Al³⁺ resistance gene from wheat, using the maize ubiquitin promoter to drive expression. TaALMT1 expression, malate efflux and Al³⁺ resistance were measured in the T₁ and T₂ lines and compared with the parental line and an Al³⁺-resistant reference genotype, ET8.ConclusionsThe Al³⁺ resistance of wheat was increased by enhancing TaALMT1 expression with biotechnology. This is the first report of a major food crop being stably transformed for greater Al³⁺ resistance. Transgenic strategies provide options for increasing food supply on acid soils.     

拟南芥AtNHX6基因启动子的克隆及表达分析

为了探明拟南芥内膜反向转运体AtNHX6基因的组织表达模式,从基因组中克隆了AtNHX6基因开放阅读框(ORF)上游侧翼调控区1 922bp序列,并成功构建AtNHX6基因启动子与GUS融合表达载体pCAM-BIA1381-proNHX6-GUS,通过农杆菌花序浸染法转化野生型拟南芥获得T3代纯合转基因拟南芥株系,经PCR检测扩增得到2 187bp目的条带。利用组织染色法鉴定转基因拟南芥的GUS表达模式发现,在子叶、下胚轴和花中GUS活性显著。在这些广泛表达的部位中,微管系统中的表达最为显著,真叶中只有局部检测到GUS表达;在根中GUS在根毛和侧根生长部位表达;在未成熟果荚中只有在果荚顶端和基部存在GUS活性,成熟果荚中只在果柄检测到GUS表达;在花中,雄蕊的花丝和花粉粒及雌蕊的柱头中检测到GUS表达。GUS染色分析结果表明,AtNHX6基因启动子与GUS的融合表达载体成功构建并正常启动GUS基因表达,且AtNHX6基因主要在拟南芥的子叶、下胚轴、根、花、果荚中的微管系统、根毛和侧根生长部位以及花丝、花粉、柱头中表达。     

Development of a simple and efficient system for excising selectable markers in Arabidopsis using a minimal promoter::Cre fusion construct

The development of rapid and efficient strategies to generate selectable marker-free transgenic plants could help increase the consumer acceptance of genetically modified (GM) plants. To produce marker-free transgenic plants without conditional treatment or the genetic crossing of offspring, we have developed a rapid and convenient DNA excision method mediated by the Cre/loxP recombination system under the control of a −46 minimal CaMV 35S promoter. The results of a transient expression assay showed that −46 minimal promoter::Cre recombinase (−46::Cre) can cause the loxP-specific excision of a selectable marker, thereby connecting the 35S promoter and β-glucuronidase (GUS) reporter gene. Analysis of stable transgenic Arabidopsis plants indicated a positive correlation between loxP-specific DNA excision and GUS expression. PCR and DNA gel-blot analysis further revealed that nine of the 10 tested T₁ transgenic lines carried both excised and nonexcised constructs in their genomes. In the subsequent T₂ generation plants, over 30% of the individuals for each line were marker-free plants harboring the excised construct only. These results demonstrate that the −46::Cre fusion construct can be efficiently and easily utilized for producing marker-free transgenic plants.     

Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins

The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, including 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tetrahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn²⁺-, Cd²⁺- or Cu⁺-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd²⁺ coordination, yielding unique Cd₁₇- and Cd₈- complexes, respectively. When binding Zn²⁺, they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu⁺, although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd²⁺ and for Cu⁺, and although not optimally, it yielded the best result when coordinating Zn²⁺. The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu₂₀-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn₁₀-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been internal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and optimization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination).     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

The AOC promoter of tomato is regulated by developmental and environmental stimuli

The allene oxide cyclase (AOC) catalyzes the formation of cis-(+)-12-oxophytodienoic acid, an intermediate in jasmonate biosynthesis and is encoded by a single copy gene in tomato. The full length AOC promoter isolated by genome walk contains 3600 bp. Transgenic tomato lines carrying a 1000 bp promoter fragment and the full length promoter, respectively, in front of the beta-glucuronidase (GUS)-encoding uidA gene and several tobacco lines carrying the full length tomato AOC promoter before GUS were used to record organ- and tissue-specific promoter activities during development and in response to various stimuli. High promoter activities corresponding to immunocytochemically detected occurrence of the AOC protein were found in seeds and young seedlings and were confined to the root tip, hypocotyl and cotyledons of 3-d-old seedlings. In 10-d-old seedlings promoter activity appeared preferentially in the elongation zone. Fully developed tomato leaves were free of AOC promoter activity, but showed high activity upon wounding locally and systemically or upon treatment with JA, systemin or glucose. Tomato flowers showed high AOC promoter activities in ovules, sepals, anthers and pollen. Most of the promoter activity patterns found in tomato with the 1000 bp promoter fragment were also detected with the full length tomato AOC promoter in tobacco during development or in response to various stimuli. The data support a spatial and temporal regulation of JA biosynthesis during development and in response to environmental stimuli.     

渗透胁迫和离子毒害对转Bt基因棉幼苗生长及离子分布的影响

研究了渗透和盐胁迫处理对转Bt基因抗虫棉(Gossypium hirsutum) 99B种子的萌发和幼苗生长的影响,以及幼苗不同器官离子吸收和分配的差异。结果表明:渗透和盐胁迫均对转Bt基因抗虫棉幼苗的生长有抑制作用,其中PEG的抑制作用最强,而3种盐的抑制程度以CaCl₂>NaCl>Na₂SO₄,且在Na⁺含量相同时,Cl^-的毒害大于SO₄^2-。渗透胁迫下使根、茎和叶中的Na⁺和Cl^-含量提高,K⁺、Ca²⁺、SO₄^2-含量和K⁺/Na⁺、Ca²⁺/Na⁺和SO₄^2-/Cl^-比值降低,且地上部的变化幅度大于地下部的,其中以PEG的影响最为显著,其次是CaCl₂,Na₂SO₄处理最弱。这些说明,转Bt基因抗虫棉99B的耐盐性较弱。     

南方根结线虫对不同砧木嫁接番茄苗活性氧清除系统的影响

采用盆栽人工接种方法,对番茄嫁接苗进行了抗性评价,研究了番茄嫁接苗叶片中抗氧化酶活性和活性氧代谢的动态变化。结果表明,接种南方根结线虫(J2)后,砧木嫁接苗表现为高抗,自根嫁接苗为高感。通过嫁接换根,与自根嫁接苗相比,砧木嫁接苗明显提高了接穗叶片的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性,降低了超氧阴离子(O.2-)产生速率以及过氧化氢(H2O2)和丙二醛(MDA)含量。表明番茄植株体内的活性氧水平和抗氧化酶活性的高低与其抗根结线虫的能力密切相关,较低的活性氧水平和较高的抗氧化酶活性有利于减轻对膜系统的伤害,提高番茄植株的抗根结线虫能力。     

紫花苜蓿对铜胁迫生理响应的傅里叶变换红外光谱法研究

为阐明紫花苜蓿(Medicago sativa L.)对铜胁迫的耐性机理,采用准确度好、分辨率高和简便快捷的傅里叶变换红外光谱法(FTIR)研究在不同铜浓度(0、1、5、20、100 mol/L)处理时紫花苜蓿根、茎、叶化学组分的变化。结果表明:随着铜处理浓度的增加,紫花苜蓿根、茎、叶生物量变化不大。其根组织在2924 cm~(-1)处峰高处呈现出先下降后上升的趋势,反映了在低铜(5 mol/L)处理条件下紫花苜蓿分泌的有机酸不断螯合Cu,造成羧酸0-H的减少,但随着Cu含量的升高,其羧酸螯合力变弱,有机酸含量渐渐升高;根组织在1381 cm~(-1)处峰高先下降后上升,反映了含油脂化合物含量先下降后升高。可能与植物在细胞壁结构上增强抗逆性有一定关系,即低Cu处理下细胞壁可能通过提高阳离子交换能力(CEC),增强了耐Cu性;茎组织在2924,1643,1381,1064 cm~(-1)等处峰高无明显变化;叶组织所有峰值在低浓度(5 mol/L)Cu处理下变化不明显,高浓度(5 moL/L)Cu处理下所有峰值先升后降,随着这可能与可溶性糖及可溶性蛋白质等物质含量都呈现先升后降的趋势有关。这表明紫花苜蓿通过根部有机酸含量的变化和提高细胞壁阳离子交换能力,将吸收的Cu大部分积累在根部,阻止Cu向地上部分运输,有效地保护了植物地上部分组织。     

A maize α-zein promoter drives an endosperm-specific expression of transgene in rice

An alpha-zein promoter isolated from maize containing P-box, E motif sequence TGTAAAGT, opaque-2 box and TATA box was studied for its tissue-specific expression in rice. A 1,098 bp promoter region of alpha-zein gene, fused to the upstream of gusA reporter gene was used for transforming rice immature embryos (ASD 16 or IR 64) via the particle bombardment-mediated method. PCR analysis of putative transformants demonstrated the presence of transgenes (the zein promoter, gusA and hpt). Nineteen out of 37 and two out of five events generated from ASD 16 and IR 64 were found to be GUS-positive. A histological staining analysis performed on sections of mature T₁ seeds revealed that the GUS expression was limited to the endosperm and not to the pericarp or the endothelial region. GUS expression was observed only in the following seed development stages : milky (14–15 DAF), soft dough (17–18 DAF), hard dough (20–23 DAF), and mature stages (28–30 DAF) of zein-gusA transformed (T₀) plants. On the contrary a constitutive expression of GUS was evident in CaMV35S-gusA plants. PCR and Southern blotting analyses on T₁ plants demonstrated a stable integration and inheritance of transgene in the subsequent T₁ generation. GUS assay on T₂ seeds revealed that the expression of gusA gene driven by alpha-zein promoter was stable and tissue-specific over two generations. Results suggest that this alpha-zein promoter could serve as an alternative promoter to drive endosperm-specific expression of transgenes in rice and other cereal transformation experiments.     

Composite Cucurbita pepo plants with transgenic roots as a tool to study root development

Background and Aims

In most plant species, initiation of lateral root primordia occurs above the elongation zone. However, in cucurbits and some other species, lateral root primordia initiation and development takes place in the apical meristem of the parental root. Composite transgenic plants obtained by Agrobacterium rhizogenes-mediated transformation are known as a suitable model to study root development. The aim of the present study was to establish this transformation technique for squash.

Methods

The auxin-responsive promoter DR5 was cloned into the binary vectors pKGW-RR-MGW and pMDC162-GFP. Incorporation of 5-ethynyl-2′-deoxyuridine (EdU) was used to evaluate the presence of DNA-synthesizing cells in the hypocotyl of squash seedlings to find out whether they were suitable for infection. Two A. rhizogenes strains, R1000 and MSU440, were used. Roots containing the respective constructs were selected based on DsRED1 or green fluorescent protein (GFP) fluorescence, and DR5::Egfp-gusA or DR5::gusA insertion, respectively, was verified by PCR. Distribution of the response to auxin was visualized by GFP fluorescence or β-glucuronidase (GUS) activity staining and confirmed by immunolocalization of GFP and GUS proteins, respectively.

Key Results

Based on the distribution of EdU-labelled cells, it was determined that 6-day-old squash seedlings were suited for inoculation by A. rhizogenes since their root pericycle and the adjacent layers contain enough proliferating cells. Agrobacterium rhizogenes R1000 proved to be the most virulent strain on squash seedlings. Squash roots containing the respective constructs did not exhibit the hairy root phenotype and were morphologically and structurally similar to wild-type roots.

Conclusions

The auxin response pattern in the root apex of squash resembled that in arabidopsis roots. Composite squash plants obtained by A. rhizogenes-mediated transformation are a good tool for the investigation of root apical meristem development and root branching.     

Efficient shoots regeneration and genetic transformation of Bacopa monniera

Bacopa monniera is an important source of metabolites with pharmaceutical value. It has been regarded as a valuable medicinal plant and its entire commercial requirement is met from wild natural population. Recently, metabolic engineering has emerged as an important solution for sustained supply of assured and quality raw material for the production of active principles. Present report describes efficient in vitro multiplication and transformation method for genetic manipulation of this species. MS medium supplemented with 2 mgl⁻¹ BA and 0.2 mgl⁻¹ IAA was found optimum for maximum shoot regeneration (98.33 %) from in vitro leaves with 2–3 longitudinal cuts. Agrobacterium tumefaciens-mediated transformation method was used for generating transgenic B. monniera plants. Putative transformants were confirmed by GUS assay and PCR based confirmation of hptII gene. DNA blot analysis showed single copy insertion of transgene cassette. An average of 87.5 % of the regenerated shoots were found PCR positive for hptII gene and GUS activity was detected in leaves of transgenic shoots at a frequency of 82.5 % The efficient multiple shoots regeneration system described herein may help in mass production of B. monniera plant. Also, the high frequency transformation protocol described here can be used for genetic engineering of B. monniera for enhancement of its pharmaceutically important metabolites.     

The barley MATE gene,HvAACT1, increases citrate efflux and Al3+ tolerance when expressed in wheat and barley

Background and Aims

Aluminium is toxic in acid soils because the soluble Al³⁺ inhibits root growth. A mechanism of Al³⁺ tolerance discovered in many plant species involves the release of organic anions from root apices. The Al³⁺-activated release of citrate from the root apices of Al³⁺-tolerant genotypes of barley is controlled by a MATE gene named HvAACT1 that encodes a citrate transport protein located on the plasma membrane. The aim of this study was to investigate whether expressing HvAACT1 with a constitutive promoter in barley and wheat can increase citrate efflux and Al³⁺ tolerance of these important cereal species.

Methods HvAACT1

was over-expressed in wheat (Triticum aestivum) and barley (Hordeum vulgare) using the maize ubiquitin promoter. Root apices of transgenic and control lines were analysed for HvAACT1 expression and organic acid efflux. The Al³⁺ tolerance of transgenic and control lines was assessed in both hydroponic solution and acid soil.

Key Results and Conclusions

Increased HvAACT1 expression in both cereal species was associated with increased citrate efflux from root apices and enhanced Al³⁺ tolerance, thus demonstrating that biotechnology can complement traditional breeding practices to increase the Al³⁺ tolerance of important crop plants.     

重金属铜、锌、镉复合胁迫对麻疯树幼苗生理生化的影响

该研究以Cu~(2+)、Zn~(2+)、Cd~(2+)单一胁迫为对照,探讨不同浓度的Cu~(2+)、Zn~(2+)、Cd~(2+)复合胁迫对麻疯树幼苗生理生化指标的影响。结果表明:随着Cu~(2+)、Zn~(2+)、Cd~(2+)浓度的增加,麻疯树幼苗叶片中的蛋白质(Pro)、丙二醛(MDA)含量均逐渐增加,其叶片叶绿素含量随着Zn~(2+)胁迫浓度的增加呈现出先降后升的趋势,在中等浓度(100 mg·L-1)的Zn~(2+)胁迫时含量最低、随着Cu~(2+)胁迫浓度的增加叶绿素含量先升高后降低,在Cu~(2+)浓度为200 mg·L-1时含量最高,达到1 200 mg·g-1FW; Cd~(2+)胁迫对叶绿素含量和根系活力无明显影响。根系活力在Zn~(2+)浓度为100 mg·L~(-1)时最强,随着Cu~(2+)浓度的增加而减弱。低浓度的Cu~(2+)、Zn~(2+)、Cd~(2+)对过氧化物酶活性和可溶性糖含量都具有促进作用。Cu~(2+)、Zn~(2+)、Cd~(2+)复合胁迫时对可溶性蛋白、叶绿素和丙二醛含量均无明显影响,随着复合胁迫时浓度的增加,可溶性糖含量和根系活力先增后减。这表明麻疯树对三种重金属的胁迫具有一定的抗性,过高浓度的胁迫会影响麻疯树幼苗生理生化的一些指标,但是麻疯树可以通过自身的防御系统使伤害降到最小。此外,重金属复合胁迫可以在一定程度上减轻单一胁迫对麻疯树幼苗造成的毒害作用。