Immunolocalization of abscisic acid by monoclonal antibodies in Lavandula stoechas L. leaves

A monoclonal antibody was used to localize abscisic acid in Lavandula stoechas L. plants treated with 1 M abscisic acid. ABA localization was performed using EDC as the only fixative, which preserves antigenicity and improves the specificity of monoclonal antibodies. A relationship was observed between the effect of abscisic acid on chloroplast ultrastructure and ABA accumulation. Gold particles accumulated on swollen chloroplasts. Our findings provide cytochemical evidence that the chloroplast is a trapping compartment, and yield valuable information on the relation between chloroplast ultrastructure and ABA accumulation.Abbreviations ABA abscisic acid - BSA bovine serum albumin - EDC 1-(3-dimethyl-aminopropyl)-3 ethyl carbodiimide - IgG immunoglobulin G - mAB monoclonal antibodies - pAB polyclonal antibodies     

Identification of AtHD2C as a novel regulator of abscisic acid responses in Arabidopsis

HD2 proteins are plant-specific histone deacetylases. Little is known about the function of HD2 proteins in plants. In this paper, we report that an Arabidopsis HD2 protein, AtHD2C, is involved in abscisic acid and abiotic stress responses. Analysis of Arabidopsis plants containing the AtHD2C:beta-glucuronidase fusion gene revealed that AtHD2C was constitutive expressed in plants. Furthermore, expression of AtHD2C was repressed by abscisic acid. Over-expression of 35S:AtHD2C-GFP in transgenic Arabidopsis plants conferred an abscisic acid-insensitive phenotype. In addition, 35S:AtHD2C-GFP transgenic plants displayed reduced transpiration and enhanced tolerance to salt and drought stresses when compared with wild-type plants. The expression of several abscisic acid-responsive genes was affected in the 35S:AtHD2C-GFP plants. Our study provides evidence indicating that AtHD2C can modulate abscisic acid and stress responses.     

Expression of a functional single-chain antibody against GA24/19 in transgenic tobacco.

An anti-gibberellin A24/19 single-chain Fv gene was constructed from gamma and kappa genes cloned from a hybridoma cell line producing monoclonal antibody against gibberellin A24/19, biosynthetic precursors of gibberellin A4/1 which are biologically active per se. The single-chain Fv gene was introduced into tobacco plants after the binding activity of the single-chain Fv expressed in Escherichia coli was confirmed. When the single-chain Fv expression is targeted to endoplasmic reticulum, the plants could accumulate the single-chain Fv protein with the antigen binding activity up to 3.6% of the total soluble protein. On the other hand, when the expression is targeted to cytosol, accumulation of the single-chain Fv protein was not detected at all. The dwarf phenotype of the transgenic plants expressing the single-chain Fv protein, together with the preliminary analytical data indicating a decreased level of gibberellin A1 in the dwarf transgenics, suggested that the single-chain Fv decreased the concentration of bioactive gibberellins by trapping and inhibiting the metabolism of gibberellin A24 and/or A19 to gibberellin A4 and/or A1.     

Immunomodulation of jasmonate to manipulate the wound response

Jasmonates are signals in plant stress responses and development. The exact mode of their action is still controversial. To modulate jasmonate levels intracellularly as well as compartment-specifically, transgenic Nicotiana tabacum plants expressing single-chain antibodies selected against the naturally occurring (3R,7R)-enantiomer of jasmonic acid (JA) were created in the cytosol and the endoplasmic reticulum. Consequently, the expression of anti-JA antibodies in planta caused JA-deficient phenotypes such as insensitivity of germinating transgenic seedlings towards methyl jasmonate and the loss of wound-induced gene expression. Results presented here suggest an essential role for cytosolic JA in the wound response of tobacco plants. The findings support the view that substrate availability takes part in regulating JA biosynthesis upon wounding. Moreover, high JA levels observed in immunomodulated plants in response to wounding suggest that tobacco plants are able to perceive a reduced level of physiologically active JA and attempt to compensate for this by increased JA accumulation.     

Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity

Expression and stability of immunoglobulins in transgenic plants have been investigated and optimized by accumulation in different cellular compartments as cytosol, apoplastic space and endoplasmic reticulum (ER) as will be discussed in this review. In several cases described the highest accumulation of complete active antibodies was achieved by targeting into the apoplastic space. High-level expression of active recombinant single-chain Fv antibodies (scFv's) was obtained by retention of these proteins in the lumen of the endoplasmic reticulum. This has been shown for leaves and seeds of transgenic tobacco as well as for potato tubers. Transgenic tobacco seeds, potato tubers and tobacco leaves can facilitate stable storage of scFv's accumulated in the ER over an extended (seeds, tubers) or a short (leaves) period of time. The expression of specific scFv's in different plant species, plant organs and cellular compartments offers the possibility of blocking regulatory factors or pathogens specifically. Examples are scFv's expressed in the cytosol and the apoplastic space of transgenic plant cells modulating the infection process of plant viruses and a cytosolically expressed scFv that influenced the activity of phytochrome A protein. The immunomodulation approach has been shown to be also applicable for investigating the action of the phyto-hormone abscisic acid (ABA). High-level accumulation of specific anti-ABA scFv's in the ER of all leaf cells has been used to block the influence of ABA on the stomatal functions. Seed-specific expression of high amounts of anti-ABA-scFv's at a defined time of seed-development induced a developmental switch from seed ripening to vegetative growth. It has been demonstrated that ER retention is essential for the accumulation of sufficient scFv to bind high concentrations of ABA in the transgenic seeds.     

Site-specific expression of single-stranded antibody fragments in Nicotiana tabacum

Antibody expression and immunomodulation are modern molecular techniques to produce pharmaceuticals and to interfere with cellular metabolism or pathogen infectivity in plants. Nonetheless, there is still no generally applicable strategy to express correctly folded active antibodies or antibody fragments in different cell compartments. To facilitate expression, single-chain antibody fragments (scFvs) were made of mouse monoclonal antibodies, J2 and P6 that specifically recognize double-stranded RNA (dsRNA). Stabilizing double-stranded replication intermediates could modulate the biological activity of dsRNAs in plants, especially to influence virus replication. Along with cytoplasmic expression, scFvs were anchored to the plasma membrane; targeted to the apoplast for secretion and made ER-resident. Expression levels were analysed and transgenic plants were evaluated for resistance or tolerance to potato virus Y infection. We have established strategies for expression of correctly assembled antibodies or antibody fragments in different plant cell compartments.     

Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen

In planta expression of recombinant antibodies recognizing pathogen-specific antigens has been proposed as a strategy for crop protection. We report the expression of fusion proteins comprising a Fusarium-specific recombinant antibody linked to one of three antifungal peptides (AFPs) as a method for protecting plants against fungal diseases. A chicken-derived single-chain antibody specific to antigens displayed on the Fusarium cell surface was isolated from a pooled immunocompetent phage display library. This recombinant antibody inhibited fungal growth in vitro when fused to any of the three AFPs. Expression of the fusion proteins in transgenic Arabidopsis thaliana plants conferred high levels of protection against Fusarium oxysporum f.sp. matthiolae, whereas plants expressing either the fungus-specific antibody or AFPs alone exhibited only moderate resistance. Our results demonstrate that antibody fusion proteins may be used as effective and versatile tools for the protection of crop plants against fungal infection.     

Prevention of stomatal closure by immunomodulation of endogenous abscisic acid and its reversion by abscisic acid treatment: physiological behaviour and morphological features of tobacco stomata

Transgenic tobacco ( Nicotiana tabacum L.) plants ubiquitously accumulating a single-chain variable-fragment (scFv) antibody against abscisic acid (ABA) to high concentrations in the endoplasmic reticulum (RA plants) show a wilty phenotype. High stomatal conductance and loss of CO(2) and light dependence of stomatal conductance are typical features of these plants. ABA was applied to these plants either via the petioles or by daily spraying over several weeks in order to normalise the phenotype. During the long-term experiments, scFv protein concentrations, total and (calculated) free ABA contents, and stomatal conductance and its dependence on CO(2) concentration and light intensity were monitored. The wilty phenotype of transgenic plants could not be normalised by short-term treatment with ABA via the petioles. Only a daily long-term treatment during plant development normalised the physiological behaviour completely. Scanning electron microscopy of stomata showed morphological changes in RA plants compared with wild-type plants that, for structural reasons, prevented regular stomatal movements. After long-term treatment with ABA this defect could be completely eliminated. Guard-cell-specific expression of the anti-ABA scFv did not cause any changes in physiological behaviour compared to the wild type. In addition, mesophyll-specific expression starting in leaves that were already fully differentiated resulted in normal phenotypes, too. We conclude that changes in distribution and availability of ABA in the cells of developing leaves of RA plants cause the development of structural features in stomata that prevent normal function.     

The Arabidopsis SUCROSE UNCOUPLED-6 gene is identical to ABSCISIC ACID INSENSITIVE-4: involvement of abscisic acid in sugar responses

In plants, sugars act as signalling molecules that control many aspects of metabolism and development. Arabidopsis plants homozygous for the recessive sucrose uncoupled-6 (sun6) mutation show a reduced sensitivity to sugars for processes such as photosynthesis, gene expression and germination. The sun6 mutant is insensitive to sugars that are substrates for hexokinase, suggesting that SUN6 might play a role in hexokinase-dependent sugar responses. The SUN6 gene was cloned by transposon tagging and analysis showed it to be identical to the previously described ABSCISIC ACID INSENSITIVE-4 (ABI4) gene. Our analysis suggests the involvement of abscisic acid and components of the abscisic acid signal transduction cascade in a hexokinase-dependent sugar response pathway. During the plant life cycle, SUN6/ABI4 may be involved in controlling metabolite availability in an abscisic acid- and sugar-dependent way.     

Abscisic Acid Content and Stomatal Sensitivity to CO(2) in Leaves of Xanthium strumarium L. after Pretreatments in Warm and Cold Growth Chambers

The degree of stomatal sensitivity to CO₂ was positively correlated with the content of abscisic acid of leaves of Xanthium strumarium grown in a greenhouse and then transferred for 24 hours or more to a cold (5/10 C, night/day) or a warm growth chamber (20/23 C). This correlation did not exist in plants kept in the greehouse continuously (high abscisic acid, no CO₂ sensitivity), nor in plants transferred from the cold to the warm chamber (low abscisic acid, high CO₂ sensitivity). The abscisic acid content of leaves was correlated with water content only within narrow limits, if at all. At equal water contents, prechilled leaves contained more abscisic acid than leaves of plants pretreated in the warm chamber. There appear to be at least two compartments for abscisic acid in the leaf.     

Expression and characterization of bispecific single-chain Fv fragments produced in transgenic plants.

We describe the expression of the bispecific antibody biscFv2429 in transgenic suspension culture cells and tobacco plants. biscFv2429 consists of two single-chain antibodies, scFv24 and scFv29, connected by the Trichoderma reesi cellobiohydrolase I linker. biscFv2429 binds two epitopes of tobacco mosaic virus (TMV): the scFv24 domain recognizes neotopes of intact virions, and the scFv29 domain recognizes a cryptotope of the TMV coat protein monomer. biscFv2429 was functionally expressed either in the cytosol (biscFv2429-cyt) or targeted to the apoplast using a murine leader peptide sequence (biscFv2429-apoplast). A third construct contained the C-terminal KDEL sequence for retention in the ER (biscFv2429-KDEL). Levels of cytoplasmic biscFv2429 expression levels were low. The highest levels of antibody expression were for apoplast-targeted biscFv2429-apoplast and ER-retained biscFv2429-KDEL that reached a maximum expression level of 1.65% total soluble protein in transgenic plants. Plant-expressed biscFv2429 retained both epitope specificities, and bispecificity and bivalency were confirmed by ELISA and surface plasmon resonance analysis. This study establishes plant cells as an expression system for bispecific single-chain antibodies for use in medical and biological applications.     

外源脱落酸对湿地植物美人蕉抗寒性的影响

低温时植物枯萎导致人工湿地运行效率降低,研究将脱落酸应用于湿地植物,以期提高其抗寒性。采用0、5、10、15、20和25 mg/L的脱落酸(ABA)对美人蕉(Canna indica L.)幼苗叶面进行喷施并进行阶段性降温培养,评价各组在0℃时的抗寒性,筛选出最适ABA处理浓度。结果表明:在低温条件下,经过适宜浓度外源脱落酸预处理组的丙二醛(MDA)、相对电导率显著性低于对照组;脯氨酸、可溶性糖和蛋白质含量显著高于对照组,超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性也有所提高。综合以上指标,认为15 mg/L脱落酸处理为最佳。随后将其应用于人工湿地,对其冬季净化效果进行评估。结果发现, 15 mg/L脱落酸预处理可使人工湿地的冬季TP、TN和COD去除率显著高于对照组(P<0.05)。由此可见,一定浓度的脱落酸预处理能够有效提高美人蕉的抗寒性,并提高人工湿地冬季净化效果。     

Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants

Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases.     

Constitutive expression of small heat shock proteins in vegetative tissues of the resurrection plant Craterostigma plantagineum

Using antibodies raised against two sunflower small heat shock proteins (sHSPs), we have detected immunologically related proteins in unstressed vegetative tissues from the resurrection plant Craterostigma plantagineum. In whole plants, further accumulation of these polypeptides was induced by heat-shock or water-stress. In desiccation-intolerant Craterostigma callus tissue, we failed to detect sHSP-related polypeptides, but their expression, and the concurrent acquisition of desiccation tolerance was induced by exogenous abscisic acid (ABA) treatment. In untressed plants, the cross-reacting polypeptides were abundant in the roots and lower part of the shoots, where they showed homogeneous tissue-distributions. This constitutive expression is novel for vegetative tissues of higher plants, and resembles the expression patterns of sHSPs in desiccation-tolerant zygotic embryos and germinating seeds.J.A. and C.A. contributed equally to this work and are both considered to be first author     

Short communication. Abscisic acid is not necessarily required for the induction of patchy stomatal closure

Patchy stomatal closure was observed in leaves of transgenic plants of Nicotiana plumbaginifolia producing antibodies that block the action of abscisic acid. Stomatal patchiness was induced by leaf detachment and subsequent water loss. Stomatal closure was followed by an irreversible reduction of maximal chlorophyll fluorescence. The degree of deviation from the A/ci-curve is correlated with steady-state diffusion conductance before leaf detachment. It is concluded that a heterogeneous sensitivity of stomata to abscisic acid is not directly involved in the induction of patchy stomatal closure.Keywords: Abscisic acid, chlorophyll fluorescence imaging, patchy stomatal closure, Nicotiana plumbaginifolia.      

Overexpression, effective renaturation, and bioactivity of novel single-chain antibodies against TNF-alpha

Neutralization of tumor necrosis factor-alpha (TNF-alpha) has become an effective therapeutic strategy for TNF-related autoimmune diseases. Due to the limitations of the large molecular inhibitors in the therapy, development of novel TNF-alpha inhibitors is very attractive and useful. In this study, based on the previously designed domain antibody, two novel human anti-TNF single-chain antibodies were constructed using modular consensus frameworks of human antibody as scaffold to display the antagonistic peptides. A variety of expression plasmids were used to determine the optimal expression system. The single-chain antibodies were always overexpressed in E.coli BL21(DE3) host as inclusion bodies. Under the optimized refolding conditions, the inclusion bodies were renatured successfully and the refolded single-chain antibodies could bind with TNF-alpha and block TNF-induced cytotoxicity on L929 cells. The bioactivity of the single-chain antibodies was significantly increased over the domain antibody.