Chimaeric genes can be constructed which fuse the transit peptide of a small subunit of the chloroplast-located ribulose 1,5-bisphosphate carboxylase with a bacterial protein. The fusion protein is translocated into chloroplasts and cleaved in a similar way to the small subunit polypeptide precursor.
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"Three cassettes that harboured a non-maize codon-optimized version (Chiu et al., 1996) were assembled under the control of either the 35S CaMV (Benfey and Chua, 1990), rice actin promoter , coupled with a 5¢-intron (Zhong et al., 1996) or the 1.7 kb maize C4 PepC promoter. (Yanagisawa and Izui, 1989) (ZmPpc1) The respective promoters were fused with translational enhancer element from the maize PPDK-A gene (Sheen, 1993), and GFP was targeted to plastids via the maize chloroplast RNA polymerase RpoTp transit peptide (Chang et al., 1999) for the 35S CaMV and rice actin cassettes, or the pea RBCS1 transit peptide (Van den Broeck et al., 1985; von Heijne et al., 1991) for the PEPC cassette. The non-codon-optimized GFP cassettes were subcloned into either the binary plasmid pPZP211 or pPZP212 (Hajdukiewicz et al., 1994), and the resultant vectors were referred to as pPTN343, pPTN372 and pPTN442, for the 35S, rice actin and PEPC promoters, respectively. "
[Show abstract][Hide abstract] ABSTRACT: Plastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll-specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath-specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon-optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid-related studies of wild-type and mutant maize plants and provide material from which different plastid types may be isolated.
"To prove this hypothesis, a protein was designed that can be targeted to both the chloroplast and the peroxisome. It is well known that the N-terminal transit peptide is necessary and sufficient for targeting a protein to the chloroplast (Van den Broeck et al., 1985; Smeekens et al., 1986; Bassham et al., 1991; Bruce, 2000; Lee et al., 2002). In contrast, the C-terminal three amino acid residues, SKL, are necessary and sufficient for targeting a protein to the peroxisome (Nito et al., 2002; Sparkes and Baker, 2002). "
[Show abstract][Hide abstract] ABSTRACT: One of the limiting factors in the production of recombinant proteins in transgenic plants is the low level of protein accumulation. A strategy was investigated for a high level of protein accumulation in plant cells. A fungal xylanase encoded by XYLII of Trichoderma reesei was chosen as the model protein because xylanases have a high potential for applications in environment-related technologies. Xylanase was expressed in the cytosol or targeted either to chloroplasts or peroxisomes alone, or to both organelles simultaneously. When xylanase was targeted to both chloroplasts and peroxisomes simultaneously the amount of xylanase accumulated was 160% of that in chloroplasts alone and 240% of that in peroxisomes alone although the transcript levels were similar among these constructs. The growth stage of the transgenic plants also affected the total amount of xylanase; the highest level of accumulation occurred at the time of flowering. This study provides genetic and biochemical data demonstrating that a high level of protein accumulation in transgenic plants can be obtained by targeting a protein to both chloroplasts and peroxisomes at the same time.
"Even very subtle changes in the vicinity of the natural cleavage site of the Rubisco small subunit precursor can lead to diminished chloroplast uptake (Wasmann et al., 1988) and/or aberrant proteolytic processing (Robinson and Ellis, 1984, 1985). It is occasionally observed that chloroplast uptake of foreign proteins can be improved by including a small portion of the mature N terminus of the transit peptide donor in addition to the transit peptide and scissile bond (Schreier et al., 1985; Van den Broeck et al., 1985). However, this approach is still associated with a high degree of unpredictability that is inextricably linked to the passenger protein. "
[Show abstract][Hide abstract] ABSTRACT: p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers. In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5' untranslated region. CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate. The leaf content of pHBA glucose conjugates in fully mature T1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight. The latter value is 50-fold higher than the best value reported for nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL. Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable. The highest CPL enzyme activity in pooled leaf material from adult T1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for nuclear transformation. These experiments demonstrate that the current limitation for pHBA production in nuclear-transformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid transformation. Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA. Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.