Expression of an Aspartate Kinase Homoserine Dehydrogenase Gene Is Subject to Specific Spatial and Temporal Regulation in Vegetative Tissues,Flowers, and Developing Seeds

Although the regulation of amino acid synthesis has been studied extensively at the biochemical level, it is still not known how genes encoding amino acid biosynthesis enzymes are regulated during plant development. In the present report, we have used the [beta]-glucuronidase (GUS) reporter gene to study the regulation of expression of an Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase (AK/HSD) gene in transgenic tobacco plants. The polypeptide encoded by the AK/HSD gene comprises two linked key enzymes in the biosynthesis of aspartate-family amino acids. AK/HSD-GUS gene expression was highly stimulated in apical and lateral meristems, lateral buds, young leaves, trichomes, vascular and cortical tissues of growing stems, tapetum and other tissues of anthers, pollen grains, various parts of the developing gynoecium, developing seeds, and, in some transgenic plants, also in stem and leaf epidermal trichomes. AK/HSD-GUS gene expression gradually dimished upon maturation of leaves, stems, floral tissues, and embryos. GUS expression was relatively low in roots. During seed development, expression of the AK/HSD gene in the embryo was coordinated with the initiation and onset of storage protein synthesis, whereas in the endosperm it was coordinated with the onset of seed desiccation. Upon germination, AK/HSD-GUS gene expression in the hypocotyl and the cotyledons was significantly affected by light. The expression pattern of the A. thaliana AK/HSD-GUS reporter gene positively correlated with the levels of aspartate-family amino acids and was also very similar to the expression pattern of the endogenous tobacco AK/HSD mRNA as determined by in situ hybridization.     

Functional analysis of a recently originating,atypical presequence: mitochondrial import and processing of GUS fusion proteins in transgenic tobacco and yeast

A gene family of at least five members encodes the tobacco mitochondrial Rieske Fe-S protein (RISP). To determine whether all five RISPs are translocated to mitochondria, fusion proteins containing the putative presequences of tobacco RISPs and Escherichia coli -glucuronidase (GUS) were expressed in transgenic tobacco, and the resultant GUS proteins were localized by cell fractionation. The aminoterminal 75 and 71 residues of RISP2 and RISP3, respectively, directed GUS import into mitochondria, where fusion protein processing occurred. The amino-terminal sequence of RISP4, which contains an atypical mitochondrial presequence, can translocate the GUS protein specifically into tobacco mitochondria with apparently low efficiency.Consistent with the proposal of a conserved mechanism for protein import in plants and fungi, the tobacco RISP3 and RISP4 presequences can direct import and processing of a GUS fusion protein in yeast mitochondria. Plant presequences, however, direct mitochondrial import in yeast less efficiently than the yeast presequence, indicating subtle differences between the plant and yeast mitochondrial import machineries. Our studies show that import of RISP4 may not require positively charged amino acid residues and an amphipathic secondary structure; however, these structural properties may improve the efficiency of mitochondrial import.     

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyl-transferase (FTase) gene was examined using transgenic expression of the β-glucuronidase (GUS) gene fused to a 3.2 kb 5′ upstream sequence of the gene encoding the pea FTase β subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase β subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.     

Chromosome locations of five genes encoding photosynthetic electron transfer proteins in Arabidopsis thaliana

The chromosome locations of nuclear genes encoding four photosynthetic electron transfer proteins have been determined by examining restriction fragment length polymorphisms in F₈recombinant inbred lines of Arabidopsis thaliana. The single-copy PetC gene encoding the chloroplast Rieske FeS protein was mapped to the top of chromosome 4, whereas the PetE and PetF genes encoding plastocyanin and ferredoxin, respectively, were mapped to different parts of chromosome 1. Two PetH genes encoding ferredoxin-NADP⁺oxidoreductase were mapped to the top of chromosome 1 and the bottom of chromosome 5.     

An Arabidopsis gene encoding a chloroplast-targeted beta-amylase

beta-Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that beta-amylases are not involved in the metabolism of chloroplast-localized transitory leaf starch. However, we have identified a novel beta-amylase gene, designated ct-Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic beta-amylases of higher plants. Expression of the ct-Bmy cDNA in E. coli confirmed that the encoded protein possesses beta-amylase activity. CT-BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT-BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT-BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct-Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks.     

A xylanase,AtXyn1, is predominantly expressed in vascular bundles,and four putative xylanase genes were identified in the Arabidopsis thaliana genome

The cDNA clone RXF12, which encodes a xylanase (EC 3.2.1.8), was isolated from Arabidopsis thaliana. The C-terminal half of the amino acid sequence of the deduced protein, named AtXyn1, showed similarity with the catalytic domain of barley xylanase X-1. The N-terminal half of AtXyn1 also contained three regions with sequences similar to cellulose-binding domains (CBDs). A xylanase assay revealed that transgenic A. thaliana plants expressing exogenous AtXyn1 fused with enhanced green fluorescent protein (EGFP) possessed approximately twice as much xylanase activity as wild-type plants. Observation by fluorescence microscopy of transgenic A. thaliana plants expressing a fusion protein of AtXyn1 and EGFP suggested that AtXyn1 is a cell wall protein. Analysis of the localization of beta-glucuronidase (GUS) activity in transgenic A. thaliana plants containing a chimeric gene with the upstream sequence of the AtXyn1 gene and the GUS gene demonstrated that the AtXyn1 gene is predominantly expressed in vascular bundles, but not in vessel cells. These data suggest that AtXyn1 is involved in the secondary cell wall metabolism of vascular bundle cells. A database search revealed that four putative xylanase genes exist in the A. thaliana genome, besides the AtXyn1 gene. Of these, two also contain several regions with sequences similar to CBDs in their N-terminal regions. Comparison of the amino acid sequences of the five xylanases suggests a possible process for their molecular evolution.     

Non-AUG translation initiation of mRNA encoding plastid-targeted phage-type RNA polymerase in Nicotiana sylvestris

A third nuclear gene encoding a bacteriophage T7-type RNA polymerase, NsRpoT-C, was isolated and characterized from Nicotiana sylvestris. The gene, NsRpoT-C, consists of 21 exons and 20 introns and encodes a polypeptide of 977 amino acid residues. The predicted NsRpoT-C protein shows the highest identity (72% amino acid identity) with Arabidopsis thaliana RpoT;3 which is a plastid-targeted protein. Surprisingly, comparison of the deduced amino acid sequence of NsRpoT-C with that of A. thaliana RpoT;3 predicted that the NsRpoT-C starts at a CUG triplet, a rare translation initiation codon. Transient expression assays in protoplasts from tobacco leaves demonstrated that the putative N-terminal transit peptide of NsRpoT-C encodes a targeting signal directing the protein into chloroplasts. This strongly suggests that NsRpoT-C functions as an RNA polymerase transcribing plastid-encoded genes. We have designated this protein NsRpoTp.     

Species-dependent expression of the hyoscyamine 6 beta-hydroxylase gene in the pericycle.

The tropane alkaloid scopolamine is synthesized in the pericycle of branch roots in certain species of the Solanaceae. The enzyme responsible for the synthesis of scopolamine from hyoscyamine is hyoscyamine 6 beta-hydroxylase (H6H). The gene for H6H was isolated from Hyoscyamus niger. It has an exon/intron organization very similar to those for ethylene-forming enzymes, suggesting a common evolutionary origin. The 827-bp 5' flanking region of the H6H gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to three solanaceous species by Agrobacterium-mediated transformation systems: H. niger and belladonna (Atropa belladonna), which have high and low levels, respectively, of H6H mRNA in the root, and tobacco (Nicotiana tabacum), which has no endogenous H6H gene. Histochemical analysis showed that GUS expression occurred in the pericycle and at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of hairy roots and plants of transgenic tobacco. In transgenic hairy roots and regenerated plants of belladonna, the root meristem was stained with GUS activity, except for a few transformants in which the vascular cylinder was also stained. These studies indicate that the cell-specific expression of the H6H gene is controlled by some genetic regulation specific to scopolamine-producing plants.