Plant Biotechnol Research Articles

The Arabidopsis Peroxisomal ABC Transporter, Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.

Correction

The Plant JournalVolume 63, Issue 2 p. 352-352 Free Access Correction First published: 08 July 2010 https://doi.org/10.1111/j.1365-313X.2010.04273.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat The authors of Shimada et al. (2010) would like to report the following error: The use of GFP as a marker was published previously by Stuitje et al. (2003) and Bensmihen et al. (2004). From this point of view, the method described in this paper is not novel. Unlike the methods described in the previous papers, this method targets the fluorescent protein to oil droplets, making it unstable following germination and allows a more quantitative analysis for the identification of homozygous lines, enhancing the utility of the approach. Bensmihen, S., To, A., Lambert, G., Kroj, T., Giraudat, J. and Parcy, F. (2004) Analysis of an activated ABI5 allele using a new selection method for transgenic Arabidopsis seeds. FEBS Lett.561, 127–131. Stuitje, A.R., Verbree, E.C., van der Linden, K.H., Mietkiewska, E.M., Nap, J.P. and Kneppers, T.J.A. (2003) Seed-expressed fluorescent proteins as versatile tools for easy (co)transformation and high-throughput functional genomics in Arabidopsis. Plant Biotechnol. J.1, 301–309. Reference Shimada, T.L., Shimada, T. and Hara-Nishimura, I. (2010) A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana. Plant J. 61, 519– 528. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume63, Issue2July 2010Pages 352-352 ReferencesRelatedInformation

Success stories in molecular farming—a brief overview

Success stories in molecular farming—a brief overview

In Planta Protein Sialylation through Overexpression of the Respective Mammalian Pathway

Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead to the generation of products with low efficacy. Here we report in vivo protein sialylation in plants, which have been shown to be well suited for the efficient generation of complex mammalian glycoproteins. This was achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. We show the transient overexpression and functional integrity of six mammalian proteins that act at various stages of the biosynthetic pathway and demonstrate their correct subcellular localization. Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain. Sialylation was at great uniformity when glycosylation mutants that lack plant-specific N-glycan residues were used as expression hosts. Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein. We report for the first time the incorporation of the entire biosynthetic pathway for protein sialylation in a multicellular organism naturally lacking sialylated glycoconjugates. Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants.

A companion protease inhibitor for the protection of cytosol‐targeted recombinant proteins in plants

We reported earlier the potential of tomato cathepsin D inhibitor (SlCDI) as an in-built stabilizing agent for the protection of recombinant proteins in transgenic plant leaf crude extracts (Plant Biotechnol J.4, 359-368). Here we document the potential of SlCDI for the in situ protection of proteins in potato leaves. Total protein assays with control and SlCDI-expressing potato lines indicated a positive impact of slcdi transgene expression on leaf protein content, with a mean relative increase of 35%-40% depending on the light regime. Out of approximately 700 proteins detected on 2-D gels, only 20 exhibited a significantly altered level on a protein-specific basis, whereas most proteins were up-regulated on a leaf fresh weight basis, albeit at variable rates. Quantitative reverse trancriptase-PCR assays for rubisco activase showed similar transcript levels in leaves of test and control lines despite protein levels increased by two- to threefold in SlCDI-expressing lines. These observations, along with the unrelated biological functions assigned to MS-identified proteins up-regulated in leaves and protease assays showing slightly increased proteasome activity in protein extracts of SlCDI-expressing lines, suggest a general, proteasome-independent protein stabilizing effect of SlCDI in planta. Transient expression assays with human alpha(1)-antichymotrypsin also showed a stabilizing effect for SlCDI on heterologous proteins, leading to net levels of the human protein increased by approximately 2.5-fold in SlCDI-expressing plants. These data illustrate, overall, the potential of SlCDI as an in vivo protein-stabilizing agent in transgenic plant systems, useful to improve protein levels and recombinant protein accumulation.

Establishment of high frequency plant regeneration system from leaf explants of Pinellia koreana via bulblets formation

Pinellia koreana K-H Tae & J-H Kim is a recently discovered Korea endemic medicinal plant species whose natural habitat is rapidly destroyed by industrial development. Described in this paper are culture conditions for high frequency plant regeneration via bulblet formation from leaf explant cultures of P. koreana. Leaf explants formed white nodular structures and off-white calluses at a frequency of 91.2% when cultured on MS medium supplemented with 2 mg/L BA and 0.5 mg/L NAA. However, the frequency of white nodular structures and off-white calluses formation was slightly decreased with an increasing concentration of NAA up to 4 mg/L, where the frequency reached 31.7%. Most petiole explants did not form white nodular structures and off-white calluses except the combination treatment of 2 mg/L BA and 2 mg/L NAA. Upon transfer onto MS basal medium, over 90% of nodular structures gave rise to numerous bulblets and developed into plantlets. Plantlets regenerated from bulblets were transplanted to potting soil and grown to maturity at a survival rate of over 95% in a growth chamber. Therefore, the in vitro plant regeneration system of P. koreana obtained in this study will be useful for mass propagation and long-term preservation of genetic resources of P. koreana. *Corresponding author Tel 042-860-4646 Fax 042-860-4677 E-mail: kimsw@kribb.re.kr Introduction Pinellia koreana K-H Tae & J-H Kim is a new plant species discovered at Chrisan in Korea (Tae and Kim, 2005). This plant differs from other plants belonging to the same genus Pinellia by having 3-foliolate leaves with bilobed lateral leaflets at maturity. The genus Pinellia is herbaceous medicinal plants belonging to the Araceae. The chemical composition of Pinellia is not well established as there are only a few reports on the occurrence of small amounts of alkaloids ephedrine in the tuber. Also a glycoprotein fraction was reported to have notable antiemetic effects (Kurata et al. 1998). And root contains toxins (presence of calcium oxylate), that are destroyed by drying or cooking. Pinellia is mostly used for indigestion, nausea, vomiting, gastritis, and ulcer (Lee and Cho 1987; Bown 1995). The genus of Pinellia consists of a few species in eastern Asia (Kitamura et al. 1980). Pinellia is propagated by tubers and bulbils. However, the yield and quality of tubers are gradually reduced over cultivation because of viral diseases. Therefore, introduction of a mass propagation system using in vitro culture technology such as development of virus-free strains and genetic manipulation of tubers is necessary to enhance the productivity of tubers. Despite its medicinal importance, few scientific studies of P. koreana have been reported. Only several in vitro culture studies have reported on the related species P. ternata including protoplast culture and plant regeneration (He et al. 1996), formation of micro-tubers and plantlet regeneration by thidiazuron (Yoo and Lim 1997), and clonal propagation by bulbils (Chen et al. 1989; Tsay et al. 1989). Also natural habitats of P. koreana decreased gradually because of industrialization. Tissue culture techniques may provide an alternative mean for its mass multiplication and ex-situ conservation of genetic resources. This study describes culture conditions for high frequency plant regeneration via bulblet formation from leaf explants cultures of P. koreana. 식물생명공학회지 제36권 제2호 J Plant Biotechnol Vol. 36, No. 2, 193-196 (2009) 194 ‧ Journal of Plant Biotechnology Figure 1. Plant regeneration from leaf explants of P. koreana via bulblets formation. A: Leaf explants; B: Off-white callus formation; C: White nodular structure formation; D: Bulblets formation from leaf-derived callus; E: Leaf and root development from bulblets; F: Regenerated plantlets transferred to soil; G: Morphological variation in leaf shape of regenerated plantlets; H: Morphological variation of plants after 2 year cultivation; I: Flowering of mature plants. Scale bars represents 1 mm (A, B, C, D and E), 2 cm (F, H) and 5 cm (G, I), respectively Materials and methods

Nucleocytoplasmic transit of human α1‐antichymotrypsin in tobacco leaf epidermal cells†

Recently, we have observed a nuclear localization for human alpha(1)-antichymotrypsin (AACT) expressed in the cytosol of transgenic Bright Yellow-2 (BY-2) tobacco cultured cells (see accompanying paper: Benchabane, M., Saint-Jore-Dupas, C., Bardor, M., Faye, L., Michaud, D. and Gomord, V. (2008a) Targeting and post-translational processing of human alpha(1)-antichymotrypsin in BY-2 tobacco cultured cells. Plant Biotechnol. J. doi: 10.1111/j.1467-7652.2008.00382.x). In the present article, we assess whether the intrinsic DNA-binding activity of AACT can explain its nuclear localization, and whether this same activity has an impact on its protease inhibitory potency and stability in planta. An engineered form of AACT with no DNA-binding activity, rAACTDeltaK, was compared with the wild-type polypeptide, rAACT, in terms of chymotrypsin inhibitory potency, stability in planta and distribution in tobacco cells. In accordance with available data reporting distinct sites for protease inhibition and DNA binding, rAACT and rAACTDeltaK showed similar antichymotrypsin activity, similar to the activity of native AACT purified from human plasma. As observed for AACT in BY-2 tobacco cells, a green fluorescent protein (GFP)-AACT fusion transiently expressed in the cytosol of tobacco leaf epidermal cells was detected mainly in the nucleus by confocal laser microscopy. By contrast, rAACTDeltaK expressed as a GFP fusion showed a balanced distribution between the cytosol and the nucleus, similar to the distribution pattern of free GFP exhibiting no DNA-binding affinity. In line with immunodetection data showing higher accumulation levels for GFP-AACT in tobacco leaf cells, rAACTDeltaK was more susceptible than rAACT to tryptic digestion in the presence of DNA. Overall, these observations suggest the following: (i) a retention effect of DNA on AACT in the nucleus; and (ii) a stabilizing effect of the AACT-DNA interaction on rAACT challenged with non-target proteases, which, possibly, may be useful in protecting this protein in plant expression platforms.

Corrigendum

Plant Biotechnology JournalVolume 7, Issue 2 p. 210-210 Open Access Corrigendum This article corrects the following: Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries Marc Gitzinger, Juliana Parsons, Ralf Reski, Martin Fussenegger, Volume 7Issue 1Plant Biotechnology Journal pages: 73-86 First Published online: October 9, 2008 First published: 08 January 2009 https://doi.org/10.1111/j.1467-7652.2008.00397.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Gitzinger M., Parsons J., Reski R. and Fussenegger M. (2009) Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries. In ‘Table 1: Expression levels of mammalian reporter constructs in Physcomitrella patens’, the unit for the ‘Expression level (supernatant)’ in line 6 for the construct ‘PCaMV35S-AMY-pA (pMG67)’, should be µmol/s/L. Table 1. : Expression levels of mammalian reporter constructs in Physcomitrella patens Expression vector Expression level (supernatant) Expression level (intra-cellular) PGTX-SEAP-pA (pSH17) 0.36 ± 0.02 µg/L 0.06 ± 0.007 µg/L PCaMV35S-SEAP-pA (pMG65) 1.02 ± 0.09 µg/L 0.19 ± 0.01 µg/L PGTX-SAMY-pA (pSH102) 9.0 ± 0.54 µmol/s*L 2.1 ± 0.02 µmol/s*L PCaMV35S-SAMY-pA (pMG66) 18.6 ± 2.3 µmol/s*L 4.1 ± 0.3 µmol/s*L PGTX-AMY-pA (pMG60) 1.65 ± 0.05 µmol/s*L 6.73 ± 0.34 µmol/s*L PCaMV35S-AMY-pA (pMG67) 2.5 ± 0.1 µmol/s*L 13.2 ± 1.23 µmol/s*L PGTX-VEGF121-pA (pSH100) 4.0 ± 0.1 ng/mL 0.42 ± 0.07 ng/mL PCaMV35S-VEGF121-pA (pMG68) 13.5 ± 0.5 ng/mL 1.1 ± 0.1 ng/mL PGTX-EPO-pA (pMG61) 48 ± 10.6 mU/mL 5.2 ± 0.9 mU/mL PCaMV35S-EPO-pA (pMG69) 159 ± 8.1 mU/mL 22 ± 0.95 mU/mL Abbreviations: AMY, Bacillus stearothermophilus-derived a-amylase; EPO, human erythropoietin; PCaMV35S, cauliflower mosaic virus promoter 35s; PGTX, synthetic promoter derived from the GTX homeodomain protein; SAMY, Bacillus stearothermophilus-derived secreted a-amylase; SEAP, human placental secreted alkaline phosphatase; VEGF121, human vascular endothelial growth factor 121. In the section 'VEGF121-based biopharmaceutical manufacturing using microencapsulated moss protoplasts’, the units of VEGF121 production were incorrect. Thus the third sentence in the second paragraph should be 'VEGF121 production reached 53 µg/L in a 9-day process, which compares well with forefront bioprocesses using moss protonema.’ In Figure 5(c), the units of the y-axis should be (ng/mL). The authors apologize for these errors. Reference Gitzinger M., Parsons J., Reski R. and Fussenegger M. (2009) Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries. Plant Biotechnol. J. 7, 73– 86 Volume7, Issue2February 2009Pages 210-210 ReferencesRelatedInformation

Corrigendum

Plant Biotechnology JournalVolume 6, Issue 8 p. 854-854 Open Access Corrigendum This article corrects the following: Low-acrylamide French fries and potato chips Caius M. Rommens, Hua Yan, Kathy Swords, Craig Richael, Jingsong Ye, Volume 6Issue 8Plant Biotechnology Journal pages: 843-853 First Published online: July 25, 2008 First published: 01 September 2008 https://doi.org/10.1111/j.1467-7652.2008.00372.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Rommens C.M., Yan H., Swords K., Richael C., Ye J. (2008) Low-acrylamide French fries and potato chips. The caption of Figure 4 “pSIM1256 lines” should read “pSIM1151 lines”. Figure 4 on page 847 should be as follows. Reference Rommens C.M., Yan H., Swords K., Richael C., Ye J. (2008) Low-acrylamide French fries and potato chips. Plant Biotechnol. J. 6, 843 – 853. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume6, Issue8October 2008Pages 854-854 ReferencesRelatedInformation

Binding and Glutathione Conjugation of Porphyrinogens by Plant Glutathione Transferases

Overexpression in Escherichia coli of a tau (U) class glutathione transferase (GST) from maize (Zea mays L.), termed ZmGSTU1, caused a reduction in heme levels and an accumulation of porphyrin precursors. This disruption was highly specific, with the expression of the closely related ZmGSTU2 or other maize GSTs having little effect. Expression in E. coli of a series of chimeric ZmGSTU1/ZmGSTU2 proteins identified domains responsible for disrupting porphyrin metabolism. In addition to known heme precursors, expression of ZmGSTU1 led to the accumulation of a novel glutathione conjugate of harderoporphyrin(ogen) (2,7,12,18-tetramethyl-3-vinylporphyrin-8,13,17-tripropionic acid). Using the related protoporphyrinogen as a substrate, conjugation could be shown to occur on one vinyl group and was actively catalyzed by the ZmGSTU. In plant transgenesis studies, the ZmGSTUs did not perturb porphyrin metabolism when expressed in the cytosol of Arabidopsis or tobacco. However, expression of a ZmGSTU1-ZmGSTU2 chimera in the chloroplasts of tobacco resulted in the accumulation of the harderoporphyrin(ogen)-glutathione conjugate observed in the expression studies in bacteria. Our results show that the well known ability of GSTs to act as ligand binding (ligandin) proteins of porphyrins in vitro results in highly specific interactions with porphyrinogen intermediates, which can be demonstrated in both plants and bacteria in vivo.

Corrigendum

Plant Biotechnology JournalVolume 6, Issue 2 p. 210-211 Open Access Corrigendum First published: 03 January 2008 https://doi.org/10.1111/j.1467-7652.2007.00312.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Dodo H. W., Konan K. N., Chen F. C., Egnin M. and Viquez O. M. (2007) Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity. The corresponding author's email should be hortense.dodo@aamu.edu. Figure 2 and its caption should be as follows: Figure 2Open in figure viewerPowerPoint Steps involved in the production of fertile transgenic peanut plants using pDK28, an Ara h 2 RNAi transformation vector. (a) Map of pDK28 binary plasmid mobilized into A. tumefaciens EHA105. (b) Germinated peanut embryos for preparation and co-culture of hypocotyl explants. (c) Selection of Kanamycin-resistant (Kr) shoots. (d) Rooting of regenerated Kr plants. (e) Transfer to soil and flowering. (f) Seeds from Kr peanut plants. In the section under ‘Regeneration of fertile transgenic peanut’, Figure 2 is cross-referenced incorrectly on page 137. The correct cross-reference should be ‘Kanamycin-sensitive plants turned white and died within 4 weeks, while kanamycin-resistant plants remained green and grew healthy (Figure 2c). A total of 59 kanamycin-resistant plants (Ara h 2-T0) were successfully rooted (Figure 2d) and transferred to pots in the Enconair growth chamber. Flowering occurred within 6 weeks following the transfer to the growth chamber (Figure 2e). Flowers were produced on both controls obtained by (i) direct peanut seed planting in soil (wild type) and (ii) tissue culture without Agrobacterium infection. The phenotype, plant growth and reproduction of the putative transgenic plants were indistinguishable from that of wild-type control. Peg elongated from the plants, and peanut pods were produced within 3–4 months (Figure 2f).’ In the section under ‘PCR and Southern hybridization confirmed the stable transformation of kanamycin-resistant peanut plants’, the second last sentence should read, ‘Analyses of the single digest (S) reveal that all the selected transgenic plants [plant #12 (19 pods), plant #23 (9 pods), plant #32 (4 pods), plant #39 (11 pods), plant #45 (7 pods), plant #55 (31 pods), plant #66 (3 pods) and plant #6 (25 pods)] carried each a single insert with a similar pattern of gene integration.’ Figure 5 should be labelled as follows: Figure 5Open in figure viewerPowerPoint Determination of Ara h 2 in peanut samples. (a) Sandwich ELISA shows the percentage of Ara h 2 in peanut total protein extracts. Numbers followed by the same letters are not statistically different at P < 0.05. (b) SDS-PAGE shows the protein profiles and (c) Western immunoblot analysis in crude peanut extracts to detect the presence of the Ara h 2 protein. Hybridization was performed using an Ara h 2 specific monoclonal antibody. About 0.5 µg purified Ara h 2 was loaded. WT, wild-type control sample; TCc, tissue culture non-transgenic control sample; (–), sample from a Southern negative plant; NT, not tested. Reference Dodo H. W., Konan K. N., Chen F. C., Egnin M. and Viquez O. M. (2007 ) Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity. Plant Biotechnol. J. 6, 135– 145. Google Scholar Volume6, Issue2February 2008Pages 210-211 FiguresReferencesRelatedInformation

Erratum

Plant Biotechnology JournalVolume 6, Issue 1 p. 103-103 Open Access Erratum This article corrects the following: Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing Atsushi Nagamatsu, Chikara Masuta, Mineo Senda, Hideyuki Matsuura, Atsushi Kasai, Jin-Sung Hong, Keisuke Kitamura, Jun Abe, Akira Kanazawa, Volume 5Issue 6Plant Biotechnology Journal pages: 778-790 First Published online: August 31, 2007 First published: 07 December 2007 https://doi.org/10.1111/j.1467-7652.2007.00307.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Nagamatsu A., Masuta C., Senda M., Matsuura H., Kasai A., Hong J.-S., Kitamura K., Abe J., and Kanazawa A. (2007) Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing. In the section under ‘Changes in isoflavone content in seeds as a consequence of VIGS of the CHS genes’, the names of the compounds were incorrect. Thus the second sentence in the paragraph should be ‘Changes in the levels of glucosides (daidzin and genistin) and malonylglucosides (malonyldaidzin and malonylgenistin) of isoflavones in the cotyledons of the seeds produced on soybean cultivar Fusanari after infection with CMV-A1:CHS7 were analysed by high-performance liquid chromatography (HPLC)’. In Figure 5(a), the β should be italics. Figure 5(a)Open in figure viewerPowerPoint In the references section, the title of Parinov et al. should be ‘Analysis of flanking sequences from Dissociation insertion line: a database for reverse genetics in Arabidopsis’. The publisher apologises for these errors. Reference Nagamatsu, A., Masuta, C., Senda, M., Matsuura, H., Kasai, A., Hong, J.-S., Kitamura, K., Abe, J. and Kanazawa, A. (2007) Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing. Plant Biotechnol. J. 5, 778– 790. Volume6, Issue1January 2008Pages 103-103 FiguresReferencesRelatedInformation

The Krüppel-associated Box Repressor Domain Can Trigger de Novo Promoter Methylation during Mouse Early Embryogenesis

The Krüppel-associated box (KRAB) domain is a transcriptional repression module responsible for the DNA binding-dependent gene silencing activity of hundreds of vertebrate zinc finger proteins. We previously exploited KRAB-mediated repression within the context of a tet repressor-KRAB fusion protein and of lentiviral vectors to create a method of external gene control. We demonstrated that with this system transcriptional silencing was fully reversible in cell culture as well as in vivo. Here we reveal that, in sharp contrast, KRAB-mediated repression results in irreversible gene silencing through promoter DNA methylation if it acts during the first few days of mouse development.

Higher Plant Plastids and Cyanobacteria Have Folate Carriers Related to Those of Trypanosomatids

Cyanobacterial and plant genomes encode proteins with some similarity to the folate and biopterin transporters of the trypanosomatid parasite Leishmania. The Synechocystis slr0642 gene product and its closest Arabidopsis homolog, the At2g32040 gene product, are representative examples. Both have 12 probable transmembrane domains, and the At2g32040 protein has a predicted chloroplast transit peptide. When expressed in Escherichia coli pabA pabB or folE, mutants, which are unable to produce or take up folates, the slr0642 protein and a modified At2g32040 protein (truncated and fused to the N terminus of slr0642) enabled growth on 5-formyltetrahydrofolate or folic acid but not on 5-formyltetrahydrofolate triglutamate, demonstrating that both proteins mediate folate monoglutamate transport. Both proteins also mediate transport of the antifolate analogs methotrexate and aminopterin, as evidenced by their ability to greatly increase the sensitivity of E. coli to these inhibitors. The full-length At2g32040 polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to the envelope of Arabidopsis chloroplasts in transient expression experiments. At2g32040 transcripts were present at similar levels in roots and aerial organs, indicating that the protein occurs in non-green plastids as well as chloroplasts. Insertional inactivation of At2g32040 significantly raised the total folate content of chloroplasts and lowered the proportion of 5-methyltetrahydrofolate but did not discernibly affect growth. These findings establish conservation of function among folate and biopterin transporter family proteins from three kingdoms of life.