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主题： Agrobacterium tumefaciens-mediated transformation [文库供求] |
 发表于 2011/10/27 19:30:00
Journal of Experimental Botany, Vol. 61, No. 5, pp. 1311–1319, 2010 doi:10.1093/jxb/erq009 Advance Access publication 11 February, 2010 This paper is available online free of all access charges (see  http://jxb.oxfordjournals.org/open_access.html ;for further details)RESEARCH PAPER Agrobacterium tumefaciens-mediated transformation of Cleome gynandra L., a C4 dicotyledon that is closely related to Arabidopsis thaliana Christine A. Newell1, Naomi J. Brown1, Zheng Liu1, Alexander Pflug2, Udo Gowik2, Peter Westhoff2 and Julian M. Hibberd1,* 1 Department of Plant Sciences, Downing Street, University of Cambridge, Cambridge CB2 3EA, UK 2 Institute of Plant Molecular and Developmental Biology, Universita¨ tsstrasse 1, Heinrich-Heine-University, D-40225 Du¨ sseldorf, Germany * To whom correspondence should be addressed: E-mail: julian.hibberd@plantsci.cam.ac.uk Received 30 September 2009； Revised 6 November 2009； Accepted 21 December 2009 Abstract In leaves of most C4 plants, the biochemistry of photosynthesis is partitioned between mesophyll and bundle sheath cells. In addition, their cell biology and development also differs from that in C3 plants. We have a poor understanding of the mechanisms that generate the cell-specific accumulation of proteins used in the C4 pathway, and there are few genes that have been shown to be important for the cell biology and development of C4 leaves. To facilitate functional analysis of C4 photosynthesis, and to enable knowledge from Arabidopsis thaliana to be translated to C4 species, an Agrobacterium tumefaciens-mediated transformation protocol was developed for the C4 species Cleome gynandra. A. tumefaciens, harbouring the binary vector SLJ1006, was used to transfer the uidA gene under the control of the CaMV 35S promoter into C. gynandra. Co-incubation of hypocotyls or cotyledons with SLJ1006 allowed efficient transfer of DNA into C. gynandra, and media that allowed callus production and then shoot regeneration were identified. Stable transformants of C. gynandra with detectable amounts of b-glucuronidase (GUS) were produced at an efficiency of 14%. When driven by the CaMV 35S promoter, GUS was visible in all leaf cells, whereas uidA translationally fused to a CgRbcS gene generated GUS accumulation specifically in bundle sheath cells. This transformation procedure is the first for an NAD-ME type C4 plant and should significantly accelerate the analysis of mechanisms underlying C4 photosynthesis. Key words: Agrobacterium tumefaciens, Arabidopsis thaliana, C4 photosynthesis, Cleome gynandra, transformation. Introduction C4 photosynthesis has evolved independently multiple times within the angiosperms and allows CO2 to be concentrated around Ribulose Bisphosphate Carboxylase Oxygenase (RuBisCO) (Sage, 2004). In tropical and subtropical regions the increased supply of CO2 to RuBisCO represses its oxygenase reaction, increases the rate of photosynthesis, and allows higher rates of growth compared to C3 species. In fact, the most productive native vegetation and domesticated crops all use the C4 pathway (Brown, 1999) and, as a consequence, it has been proposed that placing characteristics of C4 photosynthesis into crops such as rice could be used to increase yields (Matsuoka et al., 2001； Surridge, 2002； Hibberd et al., 2008). In most plants, the C4 pathway involves photosynthesis proteins being compartmented between mesophyll (M) and bundle sheath (BS) cells (Hatch, 1987； Brown et al., 2005), although single-celled C4 photosynthesis has been reported in aquatic and terrestrial plants (Bowes and Salvucci, 1984； Reiskind et al., 1989； Magnin et al., 1997； Voznesenskaya et al., 2001, 2002) as well as in a marine diatom (Reinfelder et al., 2000). In the two-celled version of the C4 pathway (Hatch, 1987), CO2 enters M cells and is converted into a 2010 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License http://creativecommons.org/licenses/bync/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from http://jxb.oxfordjournals.org/ ;by guest on October 19, 2011bicarbonate by carbonic anhydrase. Phosphoenolpyruvate carboxylase (PEPC) then combines HCO 3 with phosphoenolpyruvate to generate the C4 oxaloacetic acid (OAA), which is rapidly converted into either aspartate or malate. These C4 acids then diffuse into BS cells through abundant plasmodesmata, where one of three separate C4 acid decarboxylases releases CO2. In different lineages of C4 plants, the three distinct decarboxylases known as NADPdependent malic enzyme (NADP-ME), NAD-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK) have been co-opted into this process. In many cases one of the enzymes is believed to act as the primary decarboxylase with another fulfilling a secondary role (Wingler et al., 1999). The three carbon compound released after decarboxylation then diffuses back to the M, and in the case of pyruvate, rephosphorylation to PEP occurs, catalysed by pyruvate, orthophosphate dikinase (PPDK) (Hatch and Slack, 1968). Despite the basics of the C4 pathway having been defined for decades, there still is a relatively poor understanding of the mechanisms that generate the accumulation of proteins used in C4 photosynthesis in the M or BS cells (Brown et al., 2005). For example, although an element known as the mesophyll enhancing module1 (MEM1) has been identified in the PPC promoter of Flaveria bidentis (Gowik et al., 2004； Akyildiz et al., 2007) and gel-retardation assays showed that a region about 200 bp upstream of the maize PPDK gene generates mesophyll specific expression of GUS (Matsuoka and Numazawa, 1991), in neither case have trans-factors responsible for generating M-specific expression been isolated. In the C4 plants studied to date, RbcS seems to be regulated at multiple levels； including transcriptional (Viret et al., 1994； Purcell et al., 1995), posttranscriptional, and translational elongation (Berry et al., 1985, 1986, 1987, 1990) but again, no trans-factors have been isolated. For many of the other genes encoding enzymes (e.g. carbonic anhydrase, malate dehydrogenase, phosphoenolpyruvate carboxykinase) that have been recruited into the C4 pathway, there is no information on mechanisms generating cell-specific expression. Our understanding of the genetic basis associated with the alterations in cell biology and development of a C4 leaf is also poor. For example, there have been no genes identified to date that control the expansion, proliferation or polarized positioning of chloroplasts in BS cells, the increased plasmodesmatal connectivity between M and BS cells, nor Kranz anatomy itself. This is despite the fact that genes have been isolated in Arabidopsis thaliana that are involved in many of these processes. For example, the Min, FtsZ, and GC1 genes are known to control chloroplast division (Colletti et al., 2000； Vitha et al., 2001； Maple et al., 2004), CHUP1 is involved in controlling chloroplast movement (Oikawa et al., 2003) and glucan 1,3-b-glucosidases are implicated in plasmodesmatal connectivity (Levy et al., 2007). To date, it has not been possible to determine whether alterations to any of these genes are associated with the modifications to C4 cell biology. This is partly because transformation systems for the main NADP-ME-type C4 models, maize and Flaveria, are relatively difficult (Ishida et al., 1996； Chitty et al., 1999). To our knowledge, there are no transformation systems available for NAD-ME or PEPCK-type C4 species, and the phylogenetic distance of transformable C4 species from A. thaliana also means that direct comparison with the most widely studied model plant is unlikely to b
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