Plant Molecular Biology

We investigated the copy number of the gene for alternative oxidase (AOX) of Arabidopsis thaliana by amplification by PCR and Southern hybridization. These studies indicated that there are at least four copies of the AOX gene in Arabidopsis. We isolated genomic clones containing individual copies (designated as AOX1a, AOX1b, AOX1c and AOX2) of the AOX genes. Interestingly, two of the AOX genes (AOX1a and AOX1b) were located in tandem in a ca. 5 kb region on one of the chromosomes of Arabidopsis. Comparison between genomic and cDNA sequences of the four AOX genes showed that all AOX genes are divided by three introns and the positions of the introns in AOX1a, AOX1b, AOX1c and AOX2 are the same. We examined whether expression of Arabidopsis AOX genes, like the tobacco AOX1a gene, is enhanced by treatment with antimycin A, an inhibitor of complex III in the mitochondrial respiratory chain. We found that, in young plants, the amount of Arabidopsis AOX1a mRNA was dramatically increased by addition of antimycin A, while the transcription of the other three genes (AOX1b, AOX1c and AOX2) did not respond to antimycin A. Amplification by RT-PCR showed that AOX1a and AOX1c were expressed in all organs examined (flowers and buds, stems, rosette, and roots of 8-week old plants). In contrast, transcripts of AOX1b were detected only in the flowers and buds, and transcripts of AOX2 were detected mainly in stems, rosette and roots. These results suggested that transcriptions of the four genes for alternative oxidase of Arabidopsis are differentially regulated.

The cell cycle of eukaryotes is tightly regulated through the activity of cyclin-dependent kinases. The Arabidopsis thaliana CDKA;1 (CDC2aAt) gene is thought to encode such a protein kinase, since it is actively transcribed in proliferating tissues and can complement defects in the Schizosaccharomyces pombe cdc2 gene. We analyzed the functional structure of the CDKA;1 promoter, using fusion genes between various upstream regions of CDKA;1 and the Escherichia coli β-glucuronidase (GUS) gene. A 595 bp DNA fragment upstream from the transcription start site conferred GUS activity on developing trichomes, but not on proliferating tissues. On the other hand, another upstream fragment extending to the 5′ non-coding transcribed region gave GUS activity to both proliferating tissues and developing trichomes. Against the gl2 mutant background, GUS activity directed by the 595 bp fragment was detected in single-stalk cells, but not in giant cells without obvious polar extension growth. These results revealed that the 595 bp fragment lacks cis element(s) essential for proliferating-cell-specific promoter activity, but can direct transcription in a specific period during trichome development, which does not include cell division. This suggests that CDKA;1 functions during cell morphogenesis as well as cell proliferation.

Higher-order packaging of DNA in chromatin structures could be an essential step in the complex chain of events leading to activation/repression of eukaryotic gene expression. With the goal to investigate this aspect of transcriptional regulation of plant genes involved in symbiotic interactions between legumes and rhizobia we analyze here the molecular parameters of chromatin structure in functioning root nodules, callus and radicles of pea. Morphological intactness and the typical nucleosomal organization are preserved in purified nuclei isolated from all three sources. The calculated values of nucleosomal repeat changed from 185±5 bp in the nuclei of radicles to 168±5 bp and 195±6 bp in nodules and callus respectively. The observed changes are due to alterations in linker DNA lengths. The core histones are identical in all cases, but the subfractional composition of H1 linker histone is subjected to quantitative alterations. The most pronounced is the several-fold increase in content of the lowest-molecular-weight subfraction H1-6 which takes place during nodule development.

The precursors of the F1-ATPase β-subunits fromNicotiana plumbaginifolia andNeurospora crassa were imported into isolated spinach (Spinacia oleracea L.) leaf mitochondria. Both F1β precursors were imported and processed to mature size products. No import of the mitochondrial precursor proteins into isolated intact spinach chloroplasts was seen. Moreover, the precursor of the 33 kDa protein of photosynthetic water-splitting enzyme was not imported into the leaf mitochondria. This study provides the first experimental report ofin vitro import of precursor proteins into plant mitochondria isolated from photosynthetic tissue and enables studies of protein sorting between mitochondria and chloroplasts in a system which is homologous with respect to organelles. The results suggest a high organellar specificity in the plant cell for the cytoplasmically synthesized precursor proteins.

A photosystem I reaction center complex has been purified to homogeneity by a procedure involving partial solubilization of spinach thylakoid membranes, ion exchange chromatography and centrifugation in sucrose gradients. The complex contains 7 polypeptides: the P700 chlorophylla apoprotein with an apparent molecular weight of 67 kd, which at high resolution splits into two bands, and smaller polypeptides of 22 (subunit 2), 18.5, 18, 16, 12 and 10 kd. Stable transcripts for the P700 chlorophylla apojprotein and subunit 2 were found in plastid and cytosolic RNA, respectively. The apoprotein product obtained by translation in a mRNA-dependent cell-free rabbit reticulocyte lysate and also by DNA-programmed transcription-translation of cloned plastid DNA fragments inE. coli lysates was indistinguishable immunologically and electrophoretically from the authentic protein. However, the product immunologically related to subunit 2 was 4 kd larger than the mature compound indicating that this protein is encoded in the nucleus and synthesized as a precursor. The gene for the P700 chlorophylla apoprotein has been physically mapped on the spinach plastid chromosome by hybrid selection mapping and DNA-programmed cell-free transcription-translation using cloned restriction fragments of plastid DNA. There is one gene copy per chromosome and it is located centrally in the large single-copy region of the circular DNA molecule. This gene is uninterrupted and is transcribed in the same direction as that of the large subunit of ribulose bisphosphate carboxylase/oxygenase. Its transcript is approximately 4 kb longer than the 2 kbp structural gene.

Glucan endo-1,3-β-glucosidases (β-1,3-glucanases) have been implicated in several developmental processes and they may also play a direct role in the plant's defense against fungal pathogens. In an effort to characterize the glucanase gene family, complementary DNA clones encoding an acidic form of β-1,3-glucanase have been isolated from tobacco. The cDNA was expressed in E. coli and shown to encode a β-1,3-glucanase activity. The protein sequence encoded by the cDNA was found to match the partial protein sequence of PR-35, a previously characterized β-1,3-glucanase [29]. The protein encoded by the cDNA was purified from the extracellular fluid of TMV-infected tobacco leaves and found by immunological methods to correspond to glucanase PR-Q' [10]. From a detailed analysis of the cDNA it is clear that this glucanase represents a third structural class of enzyme which differs substantially from both the basic, vacuolar glucanase and the acidic, extracellular forms (PR-2, PR-N and PR-O). It has previously been demonstrated that the basic form of β-1,3-glucanase is synthesized as a pre-pro-enzyme and upon maturation the 21 amino acid signal peptide and a 22 amino acid carboxy-terminal peptide are removed. This processing event has been proposed to be involved with the vacuolar localization of the enzyme. By comparing the deduced protein structure of PR-Q' to that of the basic form it is evident that this extracellular enzyme is missing the carboxy-terminal 22 amino acids. The role of a conserved phenylalanine-glycine dipeptide in the processing of glucanases and other pathogenesis-related proteins from tobacco is discussed.

A full-length cDNA clone (pA23) of 832 bp encoding an oleosin from Arabidopsis thaliana was isolated by differential screening of a silique-specific cDNA library with probes prepared from poly(A)+ RNA isolated from developing seeds of wild-type (WT) Arabidopsis and from mutant AS11 with a lesion affecting diacylglycerol acyltransferase (DGAT) activity during embryo development. The encoded protein has a calculated molecular mass of 21.2 kDa, and its amino acid sequence shows strong sequence homology and structural similarity to other known oleosins. Transcription of the oleosin gene during seed development was both reduced and delayed in AS11 compared to WT. However, the level of oleosin protein did not appear to be down-regulated during seed development, and at maturity, the overall level of oleosin protein was similar in both WT and AS11. These findings indicate that regulation of oleosin gene expression is part of a highly complex, and co-ordinated expression of storage lipid biosynthesis and related (oleosin) genes during oilseed development.

Molecular evolution of the second largest subunit of low copy nuclear RNA polymerase II (RPB2) in allotetrploid StH genomic species of Elymus is characterized here. Our study first reported a 39-bp MITE stowaway element insertion in the genic region of RPB2 gene for all tetraploid Elymus St genome and diploid Pseudoroegneria spicata and P. stipifolia St genome. The sequences on 3′-end are highly conserved, with AGTA in all sequences but H10339 (E. fibrosis), in which the AGTA was replaced with AGAA. All 12 Stowaway-containing sequences encompassed a 9 bp conserved TIRs (GAGGGAGTA). Interestingly, the 5′-end sequence of GGTA which was changed to AGTA or deleted resulted in Stowaway excision in the H genome of Elymus sepcies, in which Stowaway excision did not leave footprint. Another two large insertions in all St genome sequences are also transposable-like elements detected in the genic region of RPB2 gene. Our results indicated that these three transposable element indels have occurred prior to polyploidization, and shaped the homoeologous RPB2 loci in St and H genome of Eymus species. Nucleotide diversity analysis suggested that the RPB2 sequence may evolve faster in the polyploid species than in the diploids. Higher level of polymorphism and genome-specific amplicons generated by this gene indicated that RPB2 is an excellent tool for investigating the phylogeny and evolutionary dynamics of speciation, and the mode of polyploidy formation in Elymus species.